3-Benzyl-7-triphenylmethylamino-3-cephem-4-carboxylic acid and salts and esters thereof

ABSTRACT

Substituted ceph-3-ems having a substituted amino side chain in the 7-position, carboxylic acid or a salt or ester thereof in the 4-position and a benzyl or monohalobenzyl group in the 3-position.

This is a division of application Ser. No. 303,959 filed Nov. 6, 1972,now abandoned.

This invention relates to novel substituted ceph-3-ems some of which areof value as intermediates in the synthesis of antimicrobially activecephalosporin analogues whilst others are useful as antimicrobial agentsin their own right. The invention is described in Part D of thisspecification. Parts A,B,C and E relate to starting materials andprocesses necessary for the preparation of the novel cephems.

PART A

Elsewhere we have already described a process for the preparation ofsubstituted azetidin-2-ones of formula (L) ##EQU1## wherein n represents0 or 1, X represents an amino group or a substituted amino group Rrepresents hydrogen or an organic radical; A represents a carbonyl group##EQU2## or a ketal group ##EQU3## wherein R¹ represents C₁ -C₃ alkylgroup; and B represents (i) hydrogen, (ii) a group of formula (II)##EQU4## wherein R² represents an esterified carboxylic acid group,(iii) a group of formula (III) or (IIIA) ##EQU5## wherein R³ is anesterified carboxylic acid group and R_(a), R_(b) and R_(c) are eachlower alkyl, aryl or aralkyl groups, any of which may be substituted, or(iv) a group of formula (IIIB): ##EQU6## WHEREIN R³ is an esterifiedcarboxylic acid group and R_(a) ¹ and R_(b) ¹ are substituted orunsubstituted alkoxy or aralkoxy groups; which process comprises (i)(when a compound of formula (I) wherein A is a carbonyl group is to beprepared) reacting a compound of formula (IV): ##EQU7## wherein n, X Band R are as defined with respect to formula (I) with a primary orsecondary amine and, if the resultant enamine intermediate does nothydrolyse spontaneously, subsequently subjecting the resultant enamineintermediate to acid hydrolysis to form the desired compound of formula(I) or (ii) (again when a compound of formula (I) wherein A is acarbonyl group is to be prepared) reacting a compound of formula (IV)above with water in the presence of a source of mercuric ions ascatalyst or (iii) (when a compound of formula (I) wherein A is a ketalgroup is to be prepared), reacting a compound of formula (IV) above witha lower alkanol in the presence of a source of mercuric ions ascatalyst.

The reaction of the acetylenic compound (IV) with the primary orsecondary amine under step (i) above may produce one of two possibleenamine intermediates or a mixture of both i.e. ##EQU8## In addition, itis believed that the formation of the enamines from acetylenic compounds(IV) may (at least in some cases) proceed through an intermediateallene. ##EQU9##

The process described in the preceding paragraph produces substitutedazetidin-2-ones of formula (I).

In formula (I) the group X has been defined as an amino or substitutedamino group. The term "substituted amino group" includes both mono- anddi- substituted amino groups.

Compounds of formula (I) are made from compounds of formula (IV) asstarting materials. The group X in compounds (IV) should survive thereaction conditions to end up as the group X in compound (I). Since freeamino groups or protonated amino groups tend to be somewhat reactive, itis not always desirable to carry out the reaction using compounds (IV)wherein X is either of these groups. Preferably the starting material(IV) is one wherein the group X is a substituted amino group. Theidentity of the substituents is not critical, but they should naturallybe such that the entire substituted amino group X is stable under theparticular reaction conditions chosen. If the particular substitutedamino group chosen is one which can be converted to a free amino groupwithout disruption of the β-lactam ring of compounds (I), then it may bepreferable to prepare compounds (I) wherein X is a free amino group bystarting from compound (IV) wherein X is a substituted amino group, andsubsequently removing the substituents. Examples of substituted aminogroups X which can be present in the starting materials (IV) and which,after the reaction to produce compounds (I) can be converted to freeamino groups include triphenylmethylamino (the triphenylmethyl groupbeing removable by acid hydrolysis or catalytic hydrogenation);t-butoxy-carbonylamino (removable by treatment with anhydrous acid);trichlorethoxycarbonylamino (removable by reduction with zinc and aceticacid) acylamino groups, e.g. phenylacetylamino or phenoxyacetylamino(removable, if desired, either enzymically or by known chemicalprocedures.).

Referring again to the products of the process described above, i.e. thesubstituted azetidin-2-ones (I), it will be noted that the group B ishydrogen or one of the groups (II), (III), (IIIA), or (IIIB). When B isa group of formula (II), (III), (IIIA), or (IIIB), the groups R² and R³have been defined as esterified carboxylic acid groups. Again, thisesterified carboxylic acid group takes no part in the reaction describedabove, and its identity is in this respect not critical. However, themost versatile compounds (I) are obtained when R² and R³ are esterifiedcarboxylic acid groups which can be readily converted to free carboxylicacid groups without damage to the remainder of the molecule. Examples ofsuch esters include the t-butyl and p-methoxybenzyl esters (bothremovable with a strong anhydrous acid such as trifluoroacetic acid).However, on occasions other, perhaps less readily removable esters maybe employed e.g.; lower alkyl esters or thioesters (e.g. methyl, ethylor propyl esters or thioesters); aralkyl esters or thioesters (e.g.benzyl, substituted benzyl or benzhydryl esters or thioesters); arylesters or thioesters (e.g. phenyl or substituted phenyl esters orthioesters); acyloxyalkyl esters (e.g. acetoxymethyl orpivaloyloxymethyl esters).

The group R in the starting materials of formula (IV) (and thus also inthe end products of formula (I)) has been widely defined as hydrogen oran organic group. We find that by choosing the reaction conditions andstarting materials carefully, the reaction described above can becarried out with a wide range of organic groups R present in thestarting materials. More will be said about the relationship between thegroup R and the reaction conditions later, but for the present it willbe sufficient to state that, in general, R may be hydrogen, substitutedor unsubstituted alkyl, substituted or unsubstituted aralkyl;substituted or unsubstituted aryl or a heterocyclic group which maycarry ring substituents. In particular, R may be an unsubstituted C₁ toC₆ alkyl or cycloalkyl group; a phenyl group; a phenylalkyl groupwherein the alkyl portion contains from 1 to 4 carbon atoms, an alkoxygroup having from 1 to 4 carbon atoms or a monocyclic heterocyclicgroup.

In the preceding paragraphs, reference has been made to the relationshipbetween the reaction conditions and the identity of various groups onthe starting material (IV). Before discussing this relationship in somedepth it should be noted that suitable "sources of mercuric ions" usefulin steps (ii) and (iii) of the process described above include mercuricsulphate in dilute sulphuric acid; mercuric chloride in piperidine,morpholine or pyrrolidine, mercuric acetate, mercury acetamide, mercuryp-toluene sulphonamide and a mercury-impregnated polystyrene resin inaqueous acetic acid. With water as the reactant ie. in step (ii) of theprocess described above, it is convenient to include in the reactionmixture an organic solvent for the starting material (IV) such as alower alkanol, acetic acid, acetone, dioxan, ethyl acetate, dimethylformamide, dimethyl sulphoxide or tetrahydrofuran. In general, additionof water to the triple bond occurs more readily than that of an alkanol.Hence when both water and alkanol are present the ketone (I:A =>C=O) isusually the main product although some ketal ##EQU10## may also beformed, particularly if the quantity of alkanol present greatly exceedsthat of water.

When it is desired to obtain the ketal as the major product the quantityof water present should be kept to a minimum, and it is convenient toemploy the alkanol as solvent as well as reactant. Addition of thealkanol to the triple bond of compound (IV) may be catalysed by thepreviously mentioned mercury compounds. Alternatively, a specificcatalyst which minimises the risk of hydrolysis may be formed by heatingtogether momentarily red mercuric oxide, ether-boron trifluoridecomplex, trichloroacetic acid, and the appropriate lower alkanol.

Addition of either water or lower alkanol to the triple bond may beaccomplished at temperatures between 0°C and 100°C but proceeds fasterat the higher temperatures.

It will be seen that the process described above defines two methods forthe preparation of compounds of formula (I) wherein A is the carbonylgroup. In the first, the acetylenic sulphide or sulphoxide (IV) isreacted with a primary or secondary amine to form an enamine compoundwhich then hydrolyses spontaneously to form the desired product or issubjected to acid hydrolysis to form the desired product. Sometimeshydrolysis is effected merely by subjecting the enamine compound tosilica gel chromatography. In the second, the acetylenic sulphide orsulphoxide (IV) is hydrated with water in the presence of mercuric ions.

When the sulphide or sulphoxide (IV) is reacted with a primary orsecondary amine, we find that the reaction proceeds much faster withsulphoxide (IV n=1) than with sulphide (IV n=O). Preferred aminesinclude cyclic secondary amines such as piperidine, morpholine andpyrrolidine, but other secondary amines as dimethylamine, diethylamine,dibenzylamine and primary amines such as ethylamine, n-butylamine,benzylamine, cyclohexylamine and t-butylamine may be used on occasions,especially with sulphoxides.

When the sulphides or sulphoxide (IV) is reacted with water in thepresence of the mercuric catalyst it has already been stated that theidentity of the groups X, B and R in the starting materials influencesthe choice of catalyst.

When the catalyst is mercuric sulphate/acid (in methanol, for example)the presence of acid makes it essential that the groups X and R incompounds (IV) should be acid stable. The mercury impregnatedpolystyrene resin aqueous acid appears to be useful in much the samecircumstances as HgSO₄ /H⁺, although it is less active.

When mercuric acetate, mercury acetamide, or mercury p-toluenesulphonamide is the catalyst, the groups X and R on starting material(IV) need not be acid stable, but these catalysts appear to be;effective only where R = H.

The catalyst formed by heating together momentarily red mercuric oxide,ether-boron trifluoride complex, trichloroacetic acid and theappropriate lower alkanol appears to be effective only with compounds(IV) where X is acid stable and R = H.

If desired mercuric salts may also be included in the procedure whichcomprises treating the acetylene derivative (IV) with a primary orsecondary amine to give an enamine, which in turn undergoes hydrolysisto the ketone (I A=CO). Such a procedure is typified by the use ofmercuric chloride in piperidine. In certain cases, particularly when R =H and n =O, reactions which only occur on heating when piperidine isused alone takes place at room temperature when mercuric chloride isincluded. In other cases, particularly when R is an organic radical, useof a mercury salt in combination with a primary or secondary amineappears to offer little or no advantage over the use of the amine alone.

To summarise, the most generally useful procedure for converting theacetylene derivative (IV) into the corresponding ketone (I, A=CO) isreaction with a primary or secondary amine, either alone or in thepresence of a mercury salt, followed by very mild acid hydrolysis of theintermediate enamine. This procedure is particularly advantageous when Xis an acidlabile group such as tritylamine. Addition to the triple bondoccurs faster when n = 1 than when n = O and in certain instances, aswhen R is methyl, ethyl or benzyl, only occurs at an appreciable ratewhen n = 1.

Direct addition of water to the triple bond in the presence of a mercurycompound as catalyst is chiefly useful when R = H and, if acidicconditions are employed, when X is acidstable.

In addition, HgCl.sub. 2 /piperidine is not effective with compounds(IV) where B = H. If in any case it is desired to prepare compounds (I)where n=O (i.e. the sulphide), and the process described above onlyallows the preparation of the corresponding compounds where n=1 (i.e.the sulphoxide) then the sulphoxide can be converted to the sulphide bymethods known for reducing penicillin or cephalosporin sulphoxides tothe parent penicillins and cephalosporins. Such methods are describedfor example in Belgian Pat. No. 737121.

The starting materials of formula (IV) are obtained in various ways aswill become clear from the ensuing discussion:

Compounds of formula (IV) wherein n=O, B=a group of formula (II), X is asubstituted amino group and R is defined with reference to formula (I)may be prepared by a process which comprises reacting a penicillanicacid derivative of formula (V): ##EQU11## where X is a substituted aminogroup and R² is defined with respect to formula (I) in a substantiallyanhydrous liquid medium with a strong base which does not cleave theβ-lactam ring of the penicillanic acid derivative (V) and a reagent offormula (VI):

    Z--CH.sub.2 --C .tbd. C--R                                 (VI)

wherein R is as defined with reference to formula (I) and Z is areactive atom or group of effecting the introduction of the group##EQU12## onto the sulphur atom. This reaction generally produces astarting material of formula (IV) wherein n=O, B= group of formula (II)and X and R are as defined with reference to formula (I). However, withsome bases and with some of the compounds (VI) rearrangement of themultiple bond may occur, so that the resultant product is actually amixture of starting materials (IV) and (IVA): ##EQU13## wherein n=O, b=groups of formula (II) and X and R are as defined with reference toformula (I). These two products may be identified spectroscopically and,if desired, can be separated by conventional means such aschromatography. However, it may not be necessary to isolate the isomers,since the mixture may sometimes be employed in the process of thisinvention.

Suitable strong bases which can be used to cleave the thiazolidine ringof compounds (V) include the alkali metal hydrides, particularly sodiumhydride, the alkali metal tertiary alkoxides e.g. potassium t-butoxideand the organo-derivatives of alkali metals such as sodiumdimethylsulphoxide. In general, mixtures of compounds (IV) and (IVA) aremore commonly obtained with alkali metal tertiary alkoxides than withother bases, but it is as yet impossible to generalise.

The reactive atom or group Z present in the compound (VI) may be ahalogen atom, particularly bromine or iodine. The anydrous medium inwhich the reaction is carried out may be tetrahydrofuran,dimethylformamide, dimethylsulphoxide or a mixture of t-butanol andtetrahydrofuran.

Starting materials of formula (IV) wherein n=1, B= a group of formula(II) and X and R are as defined with reference to formula (I) may beobtained by oxidation of the corresponding sulphide compound (n=O). Suchoxidiation may be carried out using the techniques known for convertingpenicillins into penicillin sulphoxides, e.g. by treatment of thesulphide with H₂ O₂ or a peracid (particularly m- chloroperbenzoicacid).

Starting materials of formula (IV) wherein n=O, B= hydrogen and X and Rare as defined in formula (I) can be prepared by treating thecorresponding compound where B= a group of formula (II) with a reagentcapable of adding oxidatively to a double bond e.g. osmium tetroxide or,preferably, potassium permanganate. This reaction removes the group (II)from the nitrogen atom of the azetidin-2-one ring and gives the desiredcompound (IV) wherein B=H. [A side rection which may take place isoxidation at the sulphur atom to form a sulphone. This side reaction canbe minimised by using mild reaction conditions]. When potassiumpermanganate is the reagent, the reaction can be carried out in varioussolvents such as acetone, aqueous acetone, pyridine and aqueouspyridine, at a temperature of from -20°C to + 10°C.

Starting materials of formula (IV) wherein n=1, B= hydrogen and X and Rare as defined with respect to formula (I) may be obtained by oxidationof the corresponding sulphide compound using H₂ O₂ or a peracid such asm-chloroperbenzoic acid.

Starting materials of formula (IV) wherein n=O B= a group of formula(III), or (IIIA) or (IIIB) and X and R are as defined may be prepared bya process which comprises reacting the corresponding compounds (IV)wherein n=O, B= hydrogen and X and R are as defined with reference toformula (I) with an ester of glyoxylic acid, thereby producing acompound of formula (VII): ##EQU14## wherein X and R are as defined withreference to formula (I) and R³ is an esterified carboxylic acid groupreacting the compound (VII) with thionyl chloride to produce a compoundof formula (VIIA): ##EQU15## wherein X, R and R³ are as already defined,and then reacting the compound of formula (VIIA) with a phosphine offormula (IIIC): ##EQU16## wherein R_(a), R_(b) and R_(c) are as definedwith reference to formula (III) or (IIIA) or a phosphine of formula(IIID): ##EQU17## wherein R_(a) ¹, R_(b) ¹ and R_(c) ¹ are substitutedor unsubstituted alkoxy or aralkoxy groups.

The first step in the above process, i.e. the reaction with the ester ofglyoxylic acid can be effected by refluxing the reaction components indry benzene with provision for continuously removing water. The secondstep, i.e. the reaction with thionyl chloride should be carried out inan inert solvent, e.g. dry tetrahydrofuran and/or dioxan in the presenceof an acid acceptor such as pyridine, under an inert atmosphere. Thionylchloride should be added to the reaction mixture in a dropwise fashion.The third step, i.e. the reaction with the phosphine (IIIC) or (IIID)should also be carried out in an inert solvent such as tetrahydrofuranand/or dioxan in the presence of an acid acceptor. Further details ofthese steps may be found in British Patent Specification No. 1,248,130.

Starting materials of formula (IV) wherein n=1, B= a group of formula(III), (IIIA) or (IIIB) and X and R are as defined may be prepared byoxidation of the appropriate compound of formula (VIIA) using, forexample H₂ O₂ or a peracid such as m-chloroperbenzoic acid, and thentreating the resultant sulphoxide with the appropriate phosphinecompound in the usual way.

PART B

The process described in Part A of this Specification permits thepreparation of a class of substituted azetidin-2-ones of formula (I):##EQU18## wherein n represents O or 1; X represents an amino orsubstituted amino group; R represents hydrogen or an organic radical; Arepresents a carbonyl group >C = O or a ketal group ##EQU19## wherein R¹represents a lower alkyl, group; and B represents (i) hydrogen, (ii) agroup of formula (II): ##EQU20## wherein R² represents an esterifiedcarboxylic acid group: (iii) a group of formula (III) or (IIIa):##EQU21## wherein R³ is an esterified carboxylic acid group and R_(a),R_(b) and R_(c) are each lower alkyl, aryl or aralkyl groups, any ofwhich may be substituted, or (IV) a group of formula (IIIB): ##EQU22##wherein R_(a) ¹ and R_(b) ¹ are substituted or unsubstituted alkoxy oraralkoxy groups.

The definitions of the symbols occurring in the above formulae (I),(II), (III), (IIIA) and (IIIB) have been dealt with in some depth inPart A of this specification, and the method for preparing the compounds(I) has also been discussed.

However, although all of the compounds of formula (I) above are usefulas intermediates in the synthesis of substituted ceph-3-ems, some areuseful at different stages in the synthesis than others. Thus, onlycompounds of formula (I) wherein X is amino or substituted amino, n=O or1, A = >C = O, R = an organic group and B = a group of formula (III),(IIIA) or (IIIB) are useful as direct precursors of substitutedceph-3-ems. The remaining compounds of formula (I) are useful forconversion to these direct precursors and are therefore intermediates atan earlier stage in the synthesis as will be apparent from the followingtypical reaction schemes. Schemes I, II and III below are illustrativeonly, and it should be understood that the order in which the varioussteps are carried out may be varied, depending on the identity of thevarious intermediates. ##SPC1##

In each of the above schemes, the individual steps have been describedgenerally in Part A of this specification. Each scheme contains a stepwhich has been represented as the reaction of the chloro- intermediate(formed after the addition of thionyl chloride) with the phosphinecompound ##EQU23## (here R_(a), R_(b) and R_(c) have the same meaning asin formula (III), (IIIA) and (IIIB)) followed by the addition of a base(the base being necessary to convert any of phosphonium compound to thedesired neutral phosphorane). If, instead of the phosphine compound##EQU24## an alternative phosphine compound ##EQU25## had been used(R_(a) ¹, R_(b) ¹ and R_(c) ¹ being alkoxy or aralkoxy groups) the finalproduct would have been one of formula:- ##SPC2##

In addition to the steps shown in Schemes I, II and III it would bepossible, should one wish to do so, to change the identity of the groupX. For example if X was originally a suitable substituted amino groupsuch as trityl it would be possible to remove the trityl group at almostany stage, to product a free amino group, which could then, if desired,be acylated (e.g. to produce the phenoxyacetylamino group) or convertedto a different substituted amino group in some other manner. In generalhowever, we prefer to retain the same group X throughout the reactionscheme.

Both reaction Schemes I, II and III above include a step whichcorresponds to the process of this invention described in Part A of thisspecification, namely hydration of the triple bond of the S-substituent.The ketal compound sometimes gives better yields after the oxidativeremoval of the isopropylidene group, than does the corresponding ketone.Ketals can, of course, be prepared in high yields from the correspondingketone and can be converted back to the ketone compound by treatmentwith acid. Thus in such cases, the group X should be acid stable.

Since they are immediate precursors of the desired ceph-3-emsstructures, a preferred class of compounds of this invention haveformula (IA) or (IB): ##EQU26## wherein n=0 or 1; X is an amino group, aprotonated amino group or a substituted amino group; R is hydrogen anorganic radical; R³ is an esterified carboxylic acid group; R_(a), R_(b)and R_(c) are each lower alkyl, aryl or aralkyl groups, any of which maybe substituted; and R_(a) ¹ and R_(b) ¹ are each substituted orunsubstituted lower alkoxy or aralkoxy groups. The compounds (IA) and(IB) can exist in several stereoisomeric forms. The preferredconfigurations is that depicted in formulae (IA¹) and (IB¹) below (N.B.:the stereo-configuration of the sulphoxides (IA¹) and (IB¹) has not beenspecified since neither isomer is especially preferred). ##EQU27##

The utility and a further description of the compounds of formula (IA)or (IB) will become apparent from the following Part C of thisspecification.

Part C

In part B above, it was said that the specific class of substitutedazetidin-2-ones having formula (IA) or (IB) above were the immediateprecursors of substituted ceph-3-ems. Such precursors are of value in aprocess for the preparation of substituted ceph-3-ems and ceph-3-emsulphoxides of formula (VIII): ##EQU28## wherein n=0 or 1, X is asubstituted amino group R³ is an esterified carboxylic acid group and Ris hydrogen or an organic radical which process comprises firstpreparing a compound of formula (IA) or (IB). ##EQU29## wherein n, X, Rand R³ are as defined with respect to formula (VIII) above, R_(a), R_(b)and R_(c) are substituted or unsubstituted lower alkyl, aryl or aralkylgroups, R_(a) ¹ and R_(b) ¹ are substituted or unsubstituted alkoxy oraralkoxy groups; and then heating said compound of formula (IA) or (IB)at a temperature of from 30°C to 150°C in an inert organic solventthereby producing the desired compound of formula (VIII). Preferably thetemperature is from 75°C to 125°C.

Suitable solvents are those which are inert under the reactionconditions and which boil between 30°C and 150°C e.g. dioxan, tolueneand benzene. High boiling solvents are difficult to remove after thereaction and are therefore not preferred. For a clean reaction, weprefer to carry the cyclisation out under an inert atmosphere, e.g. N₂,although this is not essential. In addition it is preferable to dry thesolvent thoroughly to avoid any decomposition of the starting material.

Compounds of formula (IA) or (IB) may be prepared in two ways. In thefirst method a compound of formula (IX): ##EQU30## wherein n, X, R andR³ are as defined with respect to formula (VIII) and Z is a halogenatom, or an organic sulphonyloxy group is reacted (i) with a phosphinecompound of the formula (IIIC) if a compound of formula (IA) is to beproduced: ##EQU31## wherein R_(a), R_(b) R_(c) are as defined withrespect to formula (IA), and, if necessary, a phosphonium salt compoundresulting as an intermediate product is converted to the desiredcompound of formula (IA) by elimination of the elements of the acid Hz,or (ii) with a compound of formula (IIID) if the compound of formula(IB) is to be produced: ##EQU32## wherein R_(a) ¹, R_(b) ¹ and R_(c) ¹are substituted or unsubstituted alkoxy or aralkoxy groups.

The reactive group Z in formula (IX) may be a halogen atom, preferablychlorine or bromine, or an organic sulphonyloxy group, e.g. p-toluenesulphonyloxy.

The radicals R_(a), R_(b) and R_(c) in the phosphine compound of formula(IIIC) may be optionally substituted lower alkyl or aryl (preferablyphenyl) radicals, and the radicals R_(a) ¹, R_(b) ¹ and R_(c) ¹ in thephosphine compound (IIID) may be optionally substituted lower alkoxyradicals e.g. methoxy or ethoxy, radicals.

If a phosphonium compound is obtained as an intermediate during thepreparation of compounds (IA) the elements of the acid HX may beeliminated by treatment with a weak base e.g. pyridine.

In a second method for the preparation of compounds of formula (IA) or(IB), a compound of formula (X): ##EQU33## wherein X, R and R³ are asdefined with respect to formula (VIII) and Z is as defined with respectto formula (IX), is reacted with a phosphine compound of formula (IIIC)or (IIID), and, if necessary a phosphonium salt compound resulting as anintermediate product is converted by the elimination of the elements ofthe acid HZ to a compound of formula (XI) or (XIA) as the case may be:##EQU34## X, R, R³, R_(a), R_(b), R_(c), R_(a) ¹ and R_(b) ¹ being asdefined above and the compound of formula (XI) or (XIA) is then (i)treated with a primary or secondary amine, and, if the resultant enamineintermediate does not hydrolyse spontaneously, the resultant enamineintermediate is subsequently subjected to acid hydrolysis or (ii)treated with water in the presence of a source of mercuric ions, or(iii) treated with a C₁ -C₃ alkanol in the presence of a source ofmercuric ions and an acid. Methods (ii) and (iii) are chiefly usefulwhen R=H.

It will, of course, be recognized that the process described in thepreceding paragraph is simply the process described in Part A herein,applied to a compound (XI) or (XIA), and the discussion of the reactionin Part A applies also in the present instance.

In compounds (IA) and (IB) above, the group X is a substituted aminogroup. The term "substituted amino group" includes both mono- anddi-substituted amino groups. After the cyclisation of (IA) or (IB), onheating in inert solvent the group X survives unchanged to end up in the7-position of the substituted ceph-3-em or ceph-3-em sulphoxide (VIII).Since the known antibacterially active cephalosporins have acylaminogroups in the 7-position it may sometimes be desirable that the groups Xin compounds (VIII) should be an acylamino group. This can be achievedfor example by cyclisation of compounds (IA) or (IB) wherein X is thedesired acylamino group, or (where the desired acylamino group eitherwill not survive the cyclisation step or interferes with the efficiencyof the cyclisation step) by cyclisation of compound (IA) or (IB) whereinX is a protected amino group thereby forming a compound (VIII) where Xis a protected amino group, and thereafter removing the protecting groupand acylating the free amino group by any of the methods known foracylating 7-aminocephalosporanic acid.

Examples of non-acylamino substituted amino groups X which may bepresent in compounds (IA) or (IB) and which usually survive thecyclisation step include triphenylmethylamino, t-butoxycarbonylamino andtrichloroethoxycarbonylamino. Acylamino groups which appear to survivethe cyclisation step include phenoxyacetylamino,α-(t-butoxycarbonylamino) phenylacetylamino and 2-thienylacetylaminogroups, although theoretically there is no reason why almost anyacylamino group known in the antibacterially active penicillins andcephalosporins should not be present in compounds (IA) or (IB). If thedesired acylamino group contains a reactive group such as NH₂, thisgroup may be protected during the course of the reaction.

The group R³ in compounds (IA) and (IB) is an esterified carboxylic acidgroup. Whilst almost any esterified carboxylic acid group may beemployed, we have noticed a tendency for strongly electron withdrawingesters to reduce the yield of the cyclised product (VIII). Thus, ingeneral, strongly-electron withdrawing esters such as the trichloroethylester should preferably be avoided. By analogy with the knownantibacterially active penicillins and cephalosporins, it is to beexpected that compounds of formula (VIII) wherein R³ is an esterifiedcarboxylic acid group are likely to be less active than thecorresponding compounds where R³ is free acid group or a salt of a freeacid group. Thus it is preferred that the group R³ in compounds (IA) or(IB) (which of course survives the cyclisation stage unchanged) shouldbe one which is easily converted later to a free carboxylic acid groupExamples of such esters include the t-butyl and p-methoxybenzyl esters(both removable with a strong anhydrous acid such as trifluoroaceticacid). However, on occasions other, perhaps less readily removableesters may be employed e.g.; lower alkyl esters or thioesters (e.g.methyl, ethyl or propyl esters or thioesters); aralkyl esters orthioesters (e.g. benzyl, substituted benzyl or benzhydryl esters orthioesters); aryl esters or thioesters (e.g. phenyl or substitutedphenyl esters or thioesters); acyloxyalkyl esters (e.g. acetoxymethyl orpivaloyloxymethyl esters).

The group R in compounds (IA) and (IB) above is hydrogen or an organicgroup. Since the group CH₂ R ends up in the 3-position of compounds(VIII), and since the identity of groups in the 3-position of the knowncephalosporins are known to have an effect on the antibacterial activityof the cephalosporins clearly the process of this invention is of greatimportance and versatility. It enables CH₂ R wherein R is hydrogen oralmost any organic group to be introduced at the 3-position of theceph-3-em ring where most of the previously available methods formodifying the groups at this position only allowed the substitution ofthe 3-acetoxy group of natural cephalosporins by hydrogen ornucleophilic groups. Among the organic groups R which may be present incompounds (IA) or (IB) (and therefore in compound (VIII) are alkylgroups and substituted alkyl, e.g. methyl, ethyl, n and iso-propyl, n-,sec- and tert- butyl, cyclopentyl, cyclohexyl; alkoxyalkyl groups, e.g.methoxyethyl, ethoxyethyl; acyloxy groups, e.g. acetoxy, aryl groupse.g. phenyl, naphthyl; substituted phenyl and naphthyl groups, e.g.those when the substituents are hydroxy, alkoxy, aralkoxy, carboxylicacid, salt, ester or amide derivatives of carboxylic acid groups, nitro,amino substituted amino, halogen or lower alkyl groups; aralkyl groups,e.g. benzyl, substituted benzyl, phenylethyl, substituted phenylethyl;heterocyclic groups such as tetrahydropyranyl, tetrahydropyranyloxy and2-, 3- or 4-pyridyl.

Since the cyclisation process outlined above is one step in a syntheticsequence designed to produce antibacterially active ceph-3-ems, thepreferred configuration of the starting materials (IA) and (IB) above,is that found in the naturally occurring active cephalosporins, namelythat shown in formulae (IA¹) and (IB¹) above.

Cephem sulphoxides of formula (VIII) may be reduced to cephemsthemselves by any of the conventional known methods e.g. those describedin British Pat. No. 1280693. One such method which we have foundparticularly useful is treatment with triphenylphoxphine andacetylchloride.

It will be clear from the above discussion that the cyclisation processoutlined allows the formation of a large number of substitutedceph-3-ems. Many of the compounds which can be formed by this processare esters of known cephalosporins and cephalosporin sulphoxides butsome of the compounds (VIII) are new compounds in their own right, notpreviously accessible by the known routes. The ensuing Part D of thisspecification deals with some of these new structures.

Part D

In Part C of this specification we described a process for thepreparation of some substituted ceph-3-ems and ceph-3-em sulphoxideswhich can be reduced to ceph-3-ems. Certain of these ceph-3-ems are newcompounds in their own right, some of them having antimicrobial activityand the remainder being useful as intermediates for conversion toantimicrobially active compounds.

Thus, according to the present invention there is provided a class ofsubstituted ceph-3-ems of formula (VIIIA) ##EQU35## wherein X is asubstituted amino group R³ is a carboxylic acid group or a salt or esterof a carboxylic acid group and R is a C_(alkyl) to C₁₀ aklyl orsubstituted alkyl group, a phenyl or halo- substituted phenyl group, aphenylalkyl or (halo- substituted phenyl) alkyl group having from 1 to 6carbon atoms in the alkyl portion, or a monocyclic heterocyclic groupcontaining from 5 to 7 ring atoms.

In formula (VIIIA) above X is a substituted amino group. Preferredsubstituents are those which are readily removed to leave anunsubstituted amino group, without affecting the remainder of themolecule. Examples of suitable substituted amino groups of this typeinclude triphenylmethylamino (the triphenylmethyl group being removableby catalytic hydrogenation or by treatment with acid);t-butoxycarbonylamino (removable by treatment with anhydrous acid) andtrichloroethoxycarbonylamino (removable by reduction with zinc andacetic acid).

Another preferred group of substituted amino groups include monoacylamino groups particularly those, such as phenoxyacetamido,α-amino-phenylacetamido, 2- or 3- thienylacetamido,α-azidophenylacetanido etc., which are found in the antibacteriallyactive penicillins or cephalosporins.

In formula (VIIIA) the group R may for example be ethyl, n- or iso-propyl, n-, sec, or tertbutyl, cyclopropyl, cyclobutyl, cyclohexyl,tetrahydropyranyloxymethyl, phenyl, p- fluoro-, bromo- or chloro-phenyl, benzyl, phenylethyl or tetrahydropyranyl.

Compounds of formula (VIIIA) above wherein the sterochemistry of theazetidin ring is that shown in formula (Ia¹) and wherein R³ is acarboxylic acid group or a salt thereof or an ester which is readilyhydrolysable in the body e.g. acetoxymethyl or pivaloyloxymethyl, andwherein X is an acylamino group, are usually antibacterially active. Afew of the remaining compounds of formula (IA¹) have some degree ofprincipal activity, but their princcipal use is as intermediates forconversion to antibacterially active cephalosporin analogues(substituted ceph-3-ems).

The principal novelty of the compounds of formula (I) above lies in theidentity of the group R. Until now, the range of modification whichpermitted modiification of the nucleus of the naturally occurringcephalosporins at the 3-position of the sulphur-containing ring wassomewhat limited. Compounds of formula (VIIIA) can be prepared by theprocess described in Part C of this specification, followed, wherenecessary by conversion of the carboxylic ester group in the 4-positionof the cephem ring system to the desired, carboxylic acid, or salt orester of a carboxylic acid group R³. Some of the compounds (VIIIA) maybe prepared by removing the amino-substituent(s) from the group X ofanother member of the class (VIIIA) and acylating the resultant freeamino group. This latter process is described in the ensuing Part E.

PART E

In Part D of this specification we described a class of novelsubstituted ceph-3-em derivatives which has a substituted amino group inthe 7-position of the ceph-3-em ring system. The corresponding compoundswhich have a free amino group in the 7-position of the ceph-3-em ringare useful; intermediates for the preparation of antimicrobially activecephalosporin analogues.

These useful intermediates are compounds of formula (VIIIB) and acidaddition salts thereof: ##EQU36## wherein R³ is a carboxylic acid groupor a salt or ester of a carboxylic acid group; and R is a C₂ to C₁₀alkyl or substituted alkyl group, a phenyl or halo- substituted phenylgroup, a phenylalkyl or (halo- substituted phenyl) alkyl group havingfrom 1 to 6 carbon atoms in the alkyl portion, or a monocyclicheterocyclic group containing from 5 to 7 ring atoms.

In formula (VIIIA) the group R may for example be ethyl, n- or iso-propyl, n-, sec-, or tertbutyl, cyclopropyl cyclobutyl, cyclohexyl,tetrahydropyranyloxymethyl, phenyl, p- fluoro-, bromo- or chloro-phenyl, benzyl, phenylethyl or tetrahydropyranyl.

Compounds of formula (VIIIB) may be prepared from compounds of formula(VIIIA) (described in Part D herein) by removal of the substituent fromthe substituted amino group X and, if desired, further modification ofthe group R³. The modifications which are permissible on the group R³ ofcompounds of formulae (VIIIA) and (VIIIB) will be readily apparent tothose familiar with penicillin and cephalosporin chemistry, e.g. removalof the ester group to give a free acid, conversion of the free acidgroup to a salt or new ester derivative.

The intermediates (VIIIB) are useful in a process for the preparation ofcephalosproin analogues of formula (VIIIC): ##EQU37## wherein R¹ is anorganic acyl group; R is a lower alkyl or substituted alkyl phenyl orsubstituted phenyl, benzyl or substituted benzyl group; R³ is acarboxylic acid group or a salt, ester or thioester derivative of acarboxylic acid group; which process comprises reacting a compound offormula (VIIIB) above or an acid addition salt or silyl derivativethereof with a reactive acylating derivative of the appropriate acid(XII):

    r.sup.1 oh                                                 (xii)

and, if silylated derivative of a compound of formula (VIIIB) wasemployed, removing the silyl group by alcoholysis or hydrolysis to formthe desired compound of formula (VIIIB).

By the term "silyl derivative" of compound (VIIIB) we mean the productof the reaction between compound (VIIIB) and a silylating agent such asa halotrialkyl silane, a dihalodialkylsilane, a halotrialkoxysilane, adihalodialkoxysilane or a corresponding aryl or aralkyl silane andcompounds such as hexamethyldisilazane. In general halotrialkylsilanesare preferred, especially trimethylchlorosilane. The silyl derivativesof compound (VIIIB) are extremely sensitive to moisture and hydroxyliccompounds, and after reaction with the reactive acylating derivative ofcompound (XII), the silyl group of the intermediate acylated compoundcan be removed by hydrolysis or alcoholysis.

In formulae (VIIIB)(VIIIC) and (XII) above, the group R¹ is an organicacyl group. Suitable acyl groups include those which are found on theantibacterially active penicillins and cephalosporins (including thesemi-synthetic penicillins and cephalosporins) These includephenylacetyl and 3-thienylacetyl and phenoxyacetyl.

The reaction conditions for carrying out the process of this inventionare all analogous to the conditions used in the preparation of thesemisynthetic penicillins and cephalosporins. Thus, suitable reactivederivatives of the acid (IV) include acid halides, e.g. the chloride orbromide, anhydride, mixed anhydrides and the reactive intermediatesformed from the acid and a carbodiimide or a carbonyldiimidazole.Clearly, if a reactive group such as an amino group is present in theradical R¹, (as in the case of the α-aminophenylacetyl group) this willhave to be protected during the course of the reaction. In such a case,any of the protecting groups known from the literature on the synthesisof α-aminobenzylpenicillin or α-aminobenzyl cephalosporins may beemployed.

The following Examples illustrate the present invention. In each ofthese Examples where an azetidin-2-one ring is shown the stereochemicalconfiguration of the ring is the same as that found in the naturallyoccurring antibacterially active penicillins (namely the configurationshown in formula (IA¹) above).

EXAMPLE 1 i. Preparation of t-butyl3-β-phenylethyl-7-triphenylmethylamino-3-cephem-4-carboxylate (XXIII)##SPC3##

4-(4-phenyl-2-oxobutylsulphinyl)-1-(1-t-butoxycarbonyl-1-triphenylphosphoranylidenemethyl)-3-(triphenylmethylamino)azetidin-2-one (200 mg) was refluxed in dry dioxan (5 ml) under nitrogenfor 8 hours. The mixture was evaporated to give a foam. The crude foamwas chromatographed on silica gel eluting with ethyl acetate/petroleumether mixtures to give t-butyl3-β-phenylethyl-7-triphenylmethylamino-3-cephem-4-carboxylate-1-oxide asa yellow solid (87 mg.)

ν_(max) (CHCl₃) 1785, 1720 cm⁻ ¹

This sulphoxide (87 mg) was dissolved in dimethylformamide (1 ml) andcooled to 0°C. Triphenylphosphine (74 mg) and acetyl chloride (33 mg)were added and the mixture stood at 0°-5° C for 4 hours. The mixture wasdiluted with ethyl acetate (25 ml) and washed successively with dilutesodium bicarbonate solution and brine. The dried (MgSO₄) organic layerwas evaporated to give a gum. The gum was chromatographed on silica gelto give the desired cephem (XXIII) as a solid foam (77 mg)

ν_(max) (CHCl₃) 1775 cm⁻ ¹ (β-lactam); 1715 cm⁻ ¹ (ester) δppm (CDCl₃)1.51 (5.9H); 2.4-3.2 (m.7H.1 H exchanges with D₂ O); 4.19 (d.1H. J=5Hz);4.5-4.9 (m.1H); 7.0-7.9 (Aromatics).

ii. Preparation of t-butyl3-β-phenylethyl-7-(2-thienylacetamido)-3-cephem-4-carboxylate (XXIV)##SPC4##

The cephem (XXIII) (77 mg) was dissolved in acetone (1 ml) and cooled to0°C. p-Toluene sulphonic acid monohydrate (24 mg) was added and themixture was stood at 0°C for 1 hour. The mixture was then allowed toattain room temperature and was stood at room temperature for 6 hours.The mixture was evaporated. The residue was suspended in ethyl acetate(20 ml) and shaken with saturated sodium bicarbonate solution (5 ml).The organic layer was separated and washed with brine. The dried (MgSO₄)organic layer was evaporated to give t-butyl3-β-phenylethyl-7-amino-3-cephem-4-carboxylate (XXV) contaminated withtriphenylmethanol as a solid (61 mg). ##SPC5##

The crude free base (XXV) (61 mg) was dissolved in dry methylenechloride (2 ml) and cooled to -10°C. To the stirred, cooled solution wasadded triethylamine (50 mg) and 2-thienylacetyl chloride (freshlydistilled, 30 mg). The mixture was stirred at -10°C for 15 minutes. Themixture was diluted with methylene chloride (10 ml) and washed withbrine. The dried (MgSO₄) organic layer was evaporated to give a gum.

The gum was chromatographed on silica gel eluting with ethylacetate/petroleum ether mixtures to give the desired cephem (XXIV) as asolid foam (30 mg, 49%).

ν_(max) (CHCl₃) 1785 cm⁻ ¹ (β-lactam); 1720 cm⁻ ¹ (ester); 1690 cm⁻ ¹(amide).

Molecular Ion measured at 484.1512 (C₂₅ H₂₈ N₂ O₄ S₂ requires 484.1490,Error = 4.5 ppm). Fragmentation was consistent with structure.

iii. Preparation of 3-β-phenylethyl-7-(2-thienylacetamido)-3-cephem-4-carboxylic acid (XXVI) ##SPC6##

The cephem (XXIV) (29 mg) was dissolved in trifluoroacetic acid (0.5 ml)and the solution was stood at room temperature for 30 minutes. Thesolution was evaporated and the residual gum re-evaporated from drybenzene (4 × 1 ml). The residual gum was dissolved in dry ether andevaporated to give the desired cephem carboxylic acid (XXVI) as a darkyellow solid foam (24 mg, 94%).

ν_(max) (CHCl₃) 1775 cm⁻ ¹ (β-lactam); 1675 cm⁻ ¹ (amide).

The minimum inhibitory concentrations (MIC) of this compound againstfive typical Gram-positive bacteria are tabulated below:

    Organism          MIC (μg/ml in agar)                                      ______________________________________                                        B. subtilis       0.05                                                        Staph. aureus Oxford                                                                            0.05                                                        Staph. aureus Russell                                                                           1.5                                                         haemolytic Strep. CN10                                                                          0.02                                                        Strep. pneumoniae CN33                                                                          0.15                                                        ______________________________________                                    

EXAMPLE 2 i. Preparation of Methyl3-benzyl-7-tritylamino-3-cephem-4-carboxylate.(I) ##SPC7##

1-(Methoxycarbonyl-1-triphenylphosphoranylidenemethyl)-3-(triphenylmethylamino)-4-(3-phenyl-2-oxopropylthic)azetidin-2-one(135 mg) was gently refluxed in dry dioxan (20 ml) for 60 hours undernitrogen. Evaporation of the solvent and chromatography of the residueon silica gave (I) as a white solid (66 mg). Crystallisation frommethanol yielded white plates m.p. 163°,

ν_(max) (CHCl₃) 3480.(NH), 1775 (β-lactam), 1720 (ester), 1630 (doublebond) cm⁻ ¹

δppm (CDCl₃) 2.98 (d.1H, J=10Hz, D₂ O exchanged), 3.07 (s.2H), 3.77(centre of AB quartet, J=15Hz), 3.83 (s,3H), 4.3(d, 1H, J=5 Hz), 4.71(q, 1H, J=5Hz, 10Hz, collapsing to a doublet, J=5Hz, on D₂ O exchange),7.1 - 7.7 (m, 20H).

ii. Preparation of Methyl3-benzyl-7-phenoxyacetamido-3-cephem-4-carboxylate (II) ##SPC8##

Methyl 3-benzyl-7-tritylamino-3-cephem-4-carboxylate (111 mg) wasdissolved in acetone (1.5 ml.) and the solution cooled to -20°.p-Toluene sulphonic acid (40 mg) in acetone (0.5 ml) was added dropwiseover a few minutes and the solution left at 0° for 16 hours. Thin layerchromatography (TLC) still showed some unchanged starting material waspresent. A further quantity of p-toluene sulphonic acid (10 mg) wasadded and the solution left at room temperature for 4 hours. Ethylacetate was added and the solution washed with dilute aqueous sodiumbicarbonate and brine. Evaporation of the dried organic layer affordedmethyl 3-benzyl-7-amino-3-cephem-4-carboxylate contaminated with tritylalcohol.

The crude methyl 3-benzyl-7-amino-3-cephem-4-carboxylate was dissolvedin dry dichloromethane (3ml.) and the solution cooled to -20°. Drytriethylamine (22 mg) in dichloromethane (0.5 ml) was added followed byphenoxyacetyl chloride (36 mg) in dichloromethane (0.5 ml). After 15minutes the solution was washed with water (x 2), dried and evaporated.Chromatography on silica afforded methyl3-benzyl-7-phenoxyacetamido-3-cephem-4-carboxylate (II) as a white solid(61 mg). A sample recrystallised from ethyl acetate/60°-80° petroleumether had m.p. 161-162°.

ν_(max) (Nujol) 3215, 1775, 1718, 1670, 1625 cm⁻ ¹. δ ppm (CDCl₃) 3.27(AB quartet, J= 19Hz), 3.83 (AB quartet, J= 15 Hz), 3.9 (s, 3H), 4.57(s, 2H), 5.03 (d, 1H, J= 5Hz), 5.88 1q, 1H, J= 5Hz, 10Hz), 6.8-7-5 (m,11H).

The minimum concentrations of this compound required to inhibit growthof five typical Gram-positive bacteria are tabulated below.

    Organism          M.I.C. (μg/ml)AGAR                                       ______________________________________                                        B. subtilis       1.95                                                        Staph. aureus Oxford                                                                            0.98                                                        Staph. aureus Russell                                                                           15.6                                                        haemolytic Strep. CN10                                                                          1.95                                                        Strep. pneumoniae CN33                                                                          0.98                                                        ______________________________________                                    

EXAMPLE 3 i. Preparation of t-Butyl3-benzyl-7-triphenylmethylamino-3-cephem-4-carboxylate (III) ##SPC9##

1-(1-t-Butoxycarbonyl-1-triphenylphosphoranylidenemethyl)-3-(triphenylmethylamino)-4-(3-phenyl-2-oxopropylthio)azetidin-2-one (331 mg) was refluxed in dry dioxan (15 ml) undernitrogen for 25 hours. Evaporation of the solvent and chromatography ofthe residue on silica gave (III) (141 mg).

ν_(max) (CHCl₃) 3480, 1775, 1710, 1630 cm⁻ ¹ δ ppm (CDCl₃) 1.5 (s.9H),2.95 (d.1H. J= 10Hz, D₂ O exchange), 3.00 (centre of AB quartet), 3.68(centre of AB quartet, J= 15Hz), 4.25 (d. 1H. J= 5Hz), 4.70 (q. 1H, J=5Hz, 10Hz, collapsing to a doublet, J= 5Hz, on D₂ O exchange), 7.1-7.7(m, 20H).

ii. Preparation of t-Butyl 3-Benzyl-7-[2-(thienyl)acetamido]-3-cephem-4-carboxylate (IV) ##SPC10##

t-Butyl 3-benzyl-7-triphenylmethylamino-3-cephem-4-carboxylate (929 mg)was dissolved in acetone (2 ml) and the solution cooled to -20°.p-Toluene sulphonic acid (330 mg) in acetone (2 ml) was added dropwiseover 2 - 3 minutes and the solution left at 0° for 18 hours. Thecrystalline product was filtered, washed with a little cold acetone anddried (493 mg) m.p. 175°-177°. The mother liquors were taken up in ethylacetate, washed with dilute aqueous sodium bicarbonate and brine, driedand evaporated. Chromatography on silica gave crystalline t-butyl3-benzyl-7-amino-3-cephem-4-carboxylate (V). ##SPC11##

ν_(max) (Nujol) (p-toluene sulphonate of (VI) 1778, 1720, 1640 cm⁻ ¹ν_(max) (Nujol) (V as free base) 3400, 3325, 1764, 1710, 1640 cm⁻ ¹ δppm (CDCl₃) (V as free base) 1.58 (s, 9H), 1.73 (broad singlet, 2H, D₂ Oexchanged), 3.25 (centre of AB quartet, J= 19Hz), 3.77 (centre of ABquartet, J= 15Hz) 4.7 (d, 1H, J= 5Hz), 4.95 (d, 1H, J== 5Hz), 7.3 (s,5H).

The p-toluene sulphonic acid salt of V (76 mg) was suspended in drymethylene chloride (5 ml) at -20°. Dry triethylamine (60 mg) was addedfollowed by 2-thienylacetyl chloride (25 mg). The reaction mixture waswashed with water, dried and evaporated. Chromatography on silica gaveIV which crystallised on trituration with ether (44 mg).

ν_(max) (CHCl₃) 3330, 1778, 1710, 1680, 1630 cm⁻ ¹

iii. Preparation of 3-Benzyl-7-[2-thienyl)acetamido]-3-cephem-4carboxylic acid VI ##SPC12##

t-Butyl 3-benzyl-7-[2-(thienyl)acetamido]-3-cephem-4-carboxylate (IV)(44 mg) was dissolved in anhydrous trifluoroacetic acid (0.5 ml). After1 hour at room temperature, the solvent was removed under reducedpressure, toluene was added and the mixture repeated. This was repeatedtwice more to give (VI) as a pale yellow foam.

The minimum inhibitory concentrations (MIC) of this compound required toinhibit growth of five typical Grampositive bacteria are tabulatedbelow.

    ______________________________________                                        Organism          MIC (μg/ml) Agar                                         ______________________________________                                        B. subtilis       0.2                                                         Staph. aureus Oxford                                                                            0.05                                                        Staph. aureus Russell                                                                           10                                                          β-haemolytic Strep. CN10                                                                   0.2                                                         Strep. pneumoniae CN33                                                                          0.2                                                         ______________________________________                                    

iv. Preparation of 3-Benzyl-7-[D-α-aminophenylacetamido]-3-cephem-4-carboxylic acid (VII; R=H) ##SPC13## METHOD A

N-methylmorpholine (1 microdrop) was added to sodiumN-(1-methoxycarbonylpropen-2-yl)-D-α-aminophenylacetate (182 mg) in dryacetone (3 ml) and the suspension cooled to -15°. Ethyl chloroformate(73 mg) in dry acetone (1.5 ml) was added and the mixture stirred for 30minutes. The mixture was then added to t-butyl3-benzyl-7-amino-3-cephem-4-carboxylate (212 mg) in dry acetone (4 ml)at -15°. After stirring for 1 hour with no further external cooling, thesolvent was removed under reduced pressure and the residue taken up inethyl acetate. The solution was washed with aqueous sodium bicarbonateand brine, dried and evaporated. Chromatography on silica affordedunchanged starting material (124 mg) and the N-protected derivative(VII; R = CH₃ C₁ = CHCO₂ CH₃) (115 mg).

ν_(max) 3390, 3250, 1782, 1718, 1650, 1610 cm⁻ ¹.

The latter was dissolved in anhydrous trifluoroacetic acid (2 ml) andthe solution was left at room temperature for 35 minutes. The solventwas removed under reduced pressure and toluene was added and the mixturere-evaporated. This was repeated twice more and the residue trituratedwith ether to give the triiluoroacetic acid salt of (VII; R = H) as apale yellow solid (60 mg). This was taken up in 10% aqueous methanol (5ml) at -0° and the pH adjusted to 4.5 with 10% triethylamine/methanol.The solvent was removed under reduced pressure and the residuetriturated with ether/methanol. The solid was filtered off to give (VII;R= H) (18 mg), after drying in vacuo.

ν_(max) (Nujol) 3200, 1780 sh, 1768, 1690 1600 cm⁻ ¹.

METHOD B a. Preparation of t-Butyl7β[N-butoxycarbonyl)D-α-phenylglycylamido]3-benzyl-3-cephem-4-carboxylateVIII ##SPC14##

To a solution of re-distilled methyl chloroformate (100 mg) in drytetrahydrofuran (15 ml) cooled at -10° in a carbon tetrachloridecardicebath, were added, dropwise and with stirringN-(t-butoxycarbonyl)-D-α-phenylglycine (261 mg), triethylamine (105 mg :0.14 ml) and dimethylbenzylamine (1 drop) in dry tetrahydrofuran (10 ml)over 5 minutes. Twenty-five minutes following addition, t-butyl3-benzyl-7-amino-3-cephem-4-carboxylate (330 mg; regenerated from thep-toluene sulphonate salt) in dry tetrahydrofuran (5 ml) was addeddropwise over 5 minutes. The mixture was stirred at -10° for a further 2hours. The precipitated triethylamine hydrochloride was filtered off andthe filtrate evaporated in vacuo. The residual oil was dissolved inethyl acetate for successive cold washes with water, 5% hydrochloricacid, 5% aqueous sodium bicarbonate solution, and water. The dried(MgSO₄) ethyl acetate solution was evaporated and the residuechromatographed on silica to give (VIII) as a white crystalline solid(461 mg) m.p. 152- 153° (ether/petroleum ether).

ν_(max) (Nujol) 3290, 1772, 1708, 1685, 1664 cm⁻ ¹.

δppm (CDCl₃) 1.42 (s, 9H), 1.55 (s,9H), 3.17 (centre of AB quartet, J =18Hz), 3.78 (centre of AB quartet, J = 14.5 Hz), 4.94 (d, J = 5Hz, C₆ -H), 5.22 (d,α- CH), 5.72 (q and d, 2H, C₇ - H and amide NH), 6.66 (d, J= 9Hz, amide NH), 7.17-7.45 (d, 10H).

C₃₁ H₃₇ O₆ N₃ S requires C, 64.21; H, 6.43; N, 7.25; S, 5.53. Found C,64.47; H, 6.41; N, 7.13; S, 5.63.

b. Preparation of7β-[(D-α-aminophenylacetamido]-3-benzyl-3-cephem-4-carboxylic acid (VII;R = H)

t-Butyl 7 N-(tert-butoxycarbonyl)D-α-phenylglycylamido-3-benzyl-3-cephem-4-carboxylate (VIII) (220 mg) was dissolved inanhydrous trifluoroacetic acid (7 ml). After 30 minutes at roomtemperature the solvent was evaporated in vacuo. Dry toluene (5 ml) wasadded and the solvent reevaporated. This was repeated twice more. Theresidue was triturated with ether and the solid filtered to give thetrifluoroacetic acid salt of (VII; R = H) (201 mg). Some of the latter(98 mg) was dissolved in water at 5°-10°. After 15 minutes the solidthat had separated was filtered, washed with methylene chloride andether and dried in vacuo to give (VII; R = H) (57 mg).

ν_(max) (KBr) 3415, 3180, 3020, 1758, 1682, 1580 (broad) cm⁻ ¹.

ν_(max) (Nujol) 3230, 1780, 1760 (shoulder), 1692, 1642 (sh), 1590, 1570(sh.) cm⁻ ¹.

The minimum inhibitory concentrations (MIC) of this compound required toinhibit the growth of five typical Gram-positive bacteria are tabulatedbelow:

    Organism          MIC (μg/ml in agar)                                      ______________________________________                                        B. subtilis        0.25                                                       Staph. aureus Oxford                                                                            0.1                                                         Staph. aureus Russell                                                                           2.5                                                         β-haemolytic Strep. CN10                                                                   0.1                                                         Strep. pneumoniae CN 33                                                                         1.0                                                         ______________________________________                                    

EXAMPLE 4 i. Preparation of t-Butyl3-o-fluorobenzyl-7-triphenyl-methylamino-3-cephem-4-carboxylate (IX)##SPC15##

1-(1-t-butoxycarbonyl-1-triphenylphosphoranylidenemethyl)-3-(triphenylmethylamino)-4-(3-p-fluorophenyl-2-oxopropylthio)azetidin-2-one(253 mg) was refluxed in dry dioxan (25 ml) under nitrogen for 24 hours.Evaporation of the solvent and chromatography of the residue on silicagave (IX) as a white solid (131 mg).

ν_(max) (CHCl₃) 1775, 1715 cm⁻ ¹ δ ppm (CDCl₃) 1.52 (s,3H), 3.01 (q, J =17 Hz, 2H), 2.66 -3.12 (1H, exchange D₂ O, 3.63 (q, J = 15Hz, 2H). 4.28(d, J = 4.5Hz, 1H), 4.71 (multiplet collapsing to d, J = 4.5Hz on D₂ Oexchange, 1H), 6.77-7.58 (m, aromatic).

ii. Preparation of t-Butyl3-p-fluorobenzyl-7-(2-thienylacetamido)-3-cephem-4-carboxylate (X)##SPC16##

t-Butyl 3-p-fluorobenzyl-7-triphenylmethylamino-3-cephem-4-carboxylate(IX) (72 mg) was dissolved in acetone and the solution cooled to 0°.p-Toluene sulphonic acid (25 mg) in acetone (0.5 ml) was added dropwiseover a few minutes and the solution left to warm to room temperaturewith stirring. After 2.5 hours a solid had formed but t.l.c. stillshowed some unchanged (IX) to be present. The mixture was again cooledto 0° and a further quantity of p-toluene sulphonic acid (5 mg) inacetone added. After leaving at room temperature for a further 1.5hours, the white solid was filtered off. This solid was suspended inethyl acetate and treated with saturated sodium bicarbonate solution.The organic layer was washed with brine, dried and evaporated to givet-butyl 7-amino-3-p-fluorobenzyl-3-cephem-4-carboxylate (XI) (28 mg)shown to be pure by t.l.c. ##SPC17##

The free base (XI) (80 mg) was treated with 2-thienylacetyl chloride (40mg) and triethylamine (0.05 ml) in dry methylene chloride (5 ml) at-20°. After 15 minutes the solution was washed with brine, dried andevaporated. Chromatography on silica gave the cephem (X) (50 mg) whichcrystallised on trituration with dry ether.

δ_(max) (CHCl₃) 3380, 1780, 1710, 1685 cm⁻ ¹

iii. Preparation of 3-p-fluorobenzyl-7-(2-thienylacetamido)-3-cephem-4-carboxylic acid (XII) ##SPC18##

t-Butyl 3-p-fluorobenzyl-7-(2-thienylacetamido)-3-cephem-4-carboxylate(X) (36 mg) was treated with trifluoroacetic acid (5 ml) at roomtemperature for 40 minutes. The excess trifluoroacetic acid wasdistilled off azeotropically with dry benzene to give the free acid(XII) as a foam

ν_(max) (CHCl₃) 1770, 1705, 1680 cm⁻ ¹

The minimum inhibitory concentrations (MIC) of this compound required toinhibit the growth of five typical Gram-positive bacteria are tabulatedbelow:

    Organism          MIC (μg/ml in agar)                                      ______________________________________                                        B. subtilis       0.5                                                         Staph. aureus Oxford                                                                            0.25                                                        Staph. aureus Russell                                                                           10                                                          β-haemolytic Strep. CN10                                                                   0.25                                                        Strep. pneumoniae. CN33                                                                         0.25                                                        ______________________________________                                    

iv. Preparation of t-butyl3-p-fluorobenzyl-7-carboxymethylthioacetamido-3-cephem-4-carboxylate(XIII) ##SPC19## To the free base (XI)

To t-butyl 7-amino-3-p-fluorobenzyl-3-cephem-4-carboxylate (28 mg) indry methylene chloride at 0° was added dropwise thiodiacetic anhydride(9 mg) in methylene chloride. After 20 minutes evaporation of thesolvent gave (XIII), which was obtained as an amorphous solid onre-evaporation from dry ether.

ν_(max) (CHCl₃) 1770, 1715, 1680 cm⁻ ¹

v. Preparation of3-p-fluorobenzyl-7-carboxymethylthioacetamido-3-cephem-4-carboxylate(XIV) ##SPC20##

The total crude cephem (XIII) from (iv) above was treated withtrifluoroacetic acid (3 ml) at room temperature for 1 hour. The excesstrifluoroacetic acid was distilled off azeotropically with dry benzeneto give the acid (XIV) characterised as the triethylamine salt.

ν_(max) (CHCl₃) 3280, 1770, 1670 cm⁻ ¹

The minimum inhibitory concentrations (MIC) of the free acid (XIV)required to inhibit the growth of five typical Gram-positive bacteriaare tabulated below:

    Organism          MIC (μg/ml in agar)                                      ______________________________________                                        B. subtilis       100                                                         Staph. aureus Oxford                                                                            5.0                                                         Staph. aureus Russell                                                                           50                                                          β-haemolytic Strep. CN.10                                                                  25                                                          Strep. pneumoniae. CN33                                                                         25                                                          ______________________________________                                    

EXAMPLE 5 i. Preparation of t-Butyl3-ethyl-7-triphenylmethylamino-3-cephem 4-carboxylate (XV) ##SPC21##

1-(1-t-butoxycarbonyl-1-triphenylphosphoranylidenemethyl)-3-(triphenylmethylamino)-4-(2-oxolutylthio)azetidin-2-onesulphoxide (0.31 g) was refluxed in dry dioxan (20 ml) under nitrogenfor 18 hours. Evaporation of the solvent and chromatography of theresidue on silica gave t-butyl3-ethyl-7-triphenylmethylamino-3-cephem-4-carboxylate-1-oxide (0.10) asa gum

ν_(max) (CHCl₃) 1780, 1712, 1620 cm⁻ ¹

This sulphoxide (98 mg) in dimethylformamide (5 ml) at 0° was treatedwith triphenylphosphine (95 mg) and acetyl chloride (45 mg). The mixturewas left overnight at 5°. After dilution with ethyl acetate the organicphase was washed with saturated aqueous sodium bicarbonate then brine,dried and evaporated. Chromatography on silica gave the sulphide (XV) asa white amorphous solid (53 mg).

ν_(max) (CHCl₃) 1770, 1710, 1630 cm⁻ ¹ δppm (CDCl₃) 1.07 (t, J = 7.5Hz,3H), 1.54 (s, 9H), 2.27 (q, J = 7.5Hz, 2H) 2.9 (broad, 1H exchange D₂O), 3.15 (s, 2H), 4.23 (d, J = 4.5Hz, 1H), 4.67 (broad, giving d, J =4.5Hz on D₂ O exchange, 1H), 7.13-7.6 (ar.).

ii. Preparation of t-butyl3-ethyl-7-(2-thienylacetamido)-3-cephem-4-carboxylate (XVI) ##SPC22##

t-Butyl 3-ethyl-7-triphenylmethylamino-3-cephem-4-carboxylate (53 mg)was dissolved in acetone and the solution cooled to -20°. p-Toluenesulphonic acid (21 mg) in acetone was added and the mixture warmed toroom temperature. After 1 hour crystals had formed but t.l.c. showedsome unchanged starting material to be present. The mixture wasre-cooled to -10° and a further quantity of p-toluene sulphonic acidadded (5 mg). After a further 2 hours at room temperature the solventwas removed leaving a residue containing trityl alcohol and thep-toluene sulphonate of t-butyl 3-ethyl-7-amino-3-cephem-4-carboxylate.

This residue was suspended in dry methylene chloride and triethylamine(40 mg) added. The solution obtained was cooled, -20°, and freshlydistilled 2-thienylacetyl chloride (26 mg) in methylene chloride addeddropwise with stirring, over about 5 minutes. After 15 minutes thesolution was washed with brine, dried and evaporated. Chromatography onsilica gave the cephem (XVI) (23 mg).

ν_(max) (CHCl₃) 3380, 1780, 1710, 1685, 1630 cm⁻ ¹

iii. Preparation of 3-ethyl-7-(2-thienylacetamido)-3-cephem-4-carboxylicacid (XVII) ##SPC23##

t-Butyl 3-ethyl-7-(2-thienylacetamido)-3-cephem-4-carboxylate (23 mg)was treated with trifluoroacetic acid at room temperature for 1 hour.The excess trifluoroacetic acid was distilled off azeotropically withdry benzene to give the free acid (XVII) as a gum (17 mg)

ν_(max) (CHCl₃) 1770, 1705, 1680 cm⁻ ¹

The minimum inhibitory concentrations of this compound required toinhibit the growth of five typical Gram-positive bacteria are tabulatedbelow:

    Organism          MIC (μg/ml in agar)                                      ______________________________________                                        B. subtilis       2.5                                                         Staph. aureus Oxford                                                                            2.5                                                         Staph. aureus Russell                                                                           2.5                                                         β-haemolytic Strep. CN10                                                                   2.5                                                         Strep. pneumoniae. CN33                                                                         2.5                                                         ______________________________________                                    

EXAMPLE 6 Preparation of t-Butyl3-2'-tetrahydropyranyloxyethyl)-7-triphenylmethylamino-3-cephem-4-carboxylate(XVIII) ##SPC24##

1-(1-t-butoxycarbonyl-1-triphenylphosphoranylidenemethyl)-3-(triphenylmethylamino)-4-(4-tetrahydropyranyloxy-2-oxobutylthio)azetidin-2-one(220 mg) was refluxed under nitrogen in dry dioxan (10 ml) for 47 hours.Evaporation of the solvent and chromatography of the residue on silicagave (XVIII) (102 mg)

ν_(max) (CHCl₃) 1770, 1708, 1625 cm⁻ ¹

EXAMPLE 7 i. Preparation of tert-butyl3-(2-tetrahydropyranylmethyl)-7-triphenylmethylamino-3-cephem-4-carboxylate(XIX) ##SPC25##

1(1-t-butoxycarbonyl-1-triphenylphosphoranylidenemethyl)-3-triphenylmethylamino)-4-[3-(2-tetrahydropyranyl)-prop-2-onethio]azetidin-2-one(700 mg) was taken up in dioxan (20 ml) and refluxed under nitrogen for26 hours. The dioxan was then removed and the crude product waschromatographed on silica gel, eluting with 60 petroleum (b.p.60°-80°)/ethyl acetate in 8:2, 7:3 and 6:4 mixtures. Early fractionscontained the cephem (XIX) (180 mg)

ν_(max) (CHCl₃) 1770, 1710, 1625 cm⁻ ¹

Later fractions contained recovered starting material. This was againdissolved in dioxan and refluxed for three days and yielded the cephem(XIX) (70 mg).

ii. Preparation of t-butyl3-(2-tetrahydropyranylmethyl)-7-(2-thienylacetamido)-3-cephem-4-carboxylate(XX) ##SPC26##

The triphenylmethylaminocephem XIX (260 mg) was dissolved in a smallquantity of acetone (ca. 3 ml) and cooled to ca. -50°. p-Toluenesulphonic acid monohydrate (91 mg) in the minimum quantity of acetone(ca. 0.75 ml) was then added, and the mixture was left to stand at -10°for 18 hr, and then at room temperature for 3 hr. The acetone wasevaporated off and the residue was taken up in ethyl acetate and washedwith aqueous sodium bicarbonate, followed by brine. After drying (MgSO₄)the ethyl acetate was removed and the residue was chromatographed onsilica gel, eluting with chloroform, followed by 7:3 and 3:7 mixtures ofpetroleum (b.p. 60°-80°) and ethyl acetate. This led to the isolation oft-butyl 3-(2-tetrahydropyranylmethyl)-7-amino-3-cephem-4 carboxylate(XXI) (120 mg) ν_(max) (CHCl₃) 3,300 cm⁻ ¹ (--NH₂), 1775 cm⁻ ¹ (β-lactamC=0), 1710 cm⁻ ¹ (α,β-unsaturated ester C=0), 1620 cm⁻ ¹ (C=C).##SPC27##

The free base (XXI) (120 mg) was taken up in methylene chloride (5 ml),cooled to -13° and triethylamine (0.13 ml), followed by freshlydistilled 2-thienyl-acetyl chloride (100 mg) in methylene chloride (1ml), was added to the solution. The cooled mixture was stirred for 15minutes, brine was then added, the layers were separated, and theorganic layer dried (MgSO₄) and evaporated to leave an oil.Chromatography on silica gel, eluting with petroleum (b.p.60°-80°)/ethyl acetate (8:2 then 7:3, led to the isolation of thethienylacetamidocephen (XX) as an oil. ν_(max) (CHCl₃) 3325 cm⁻ ¹ (NH),1775 cm⁻ ¹ (β-lactam C=0), 1710 cm⁻ ¹ (α,β-unsaturated ester C=O), 1680cm⁻ ¹ (amido C=0). Trituration of the oil with ether gave the cephem (X)as a white solid (29 mg), m.p. 175°-185° decomp. M⁺ m/e 478 (parentpeak) and the fragmentations expected for the cephem (X).

iii. Preparation of3-(2-tetrahydropyranylmethyl)-7-(2-thienylacetamido)-3-cephem-4-carboxylicacid (XXII) ##SPC28##

The cephem ester (XX) (28 mg) was dissolved in trifluoroacetic acid (0.8ml) and allowed to stand at room temperature for 30 min. The reactionmixture was evaporated azeotropically from dry benzene to give the acid(XXII) as a glass (25 mg)

ν_(max) (CHCl₃) 1775, 1730-1750 , 1675 cm⁻ ¹

The minimum inhibitory concentrations (MIC) of this compound againstfive typical Gram-positive bacteria are tabulated below:

    Organism          MIC (μg/ml in agar)                                      ______________________________________                                        B. subtilis       0.5                                                         Staph. aureus Oxford                                                                            0.25                                                        Staph. aureus Russell                                                                           10                                                          β-haemolytic Strep. CN10                                                                   0.25                                                        Strep. pneumoniae. CN33                                                                         0.25                                                        ______________________________________                                    

Preparation of starting material for Example 1

i. Benzyl 6-β-(triphenylmethylamino)penicillanate (1.64g) was stirred indry tetrahydrofuran (60ml) containing 1-bromo-4-phenylbut-2-yne (0.69g.1.1eq., obtained by treating the corresponding hydroxy compound withPBr₃) under N₂. A 0.778M solution of potassium t-butoxide in t-butanol(4.3 ml. diluted with 15ml tetrahydrofuran) was added over 45 minutesand stirring was continued for 1.5/2 hours. The work-up was carried outas before and gave a chromatographical purified1-(1-benzyloxycarbonyl-2-methyl-1-propenyl)-3-triphenylmethylamino-4-(4-phenylbut-2-ynylthio)azetidin-2-one639mg (32%). v_(max) (CHCl₃) 1755 cm⁻ ¹ (β-lactam), 1718 cm⁻ ¹ (ester).δ ppm (CDCl₃) 1.99 (s, 3H), 2.19 (s, 3H), 2.75 (m, 3H, one H exchange),3.50 (t, 2H, J=2Hz), 4.50 (m, 1H, collapsing to doublet J=5 Hz with D₂O), 4.81 (d, 1H, J=5Hz), 5.01 (q, 2H) 7.0-7.8 (aromatic

ii.1-(1-Benzyloxycarbonyl-2-methyl-1-propenyl)-3-(triphenylmethylamino)-4-(4-phenylbut-2-ynylthio)azetidin-2-one(5.40g) was dissolved in a mixture of pyridine (60ml) and water (6ml).The stirred mixture was cooled in an ice-salt bath and finely powderedpotassium permanganate (2.30g) was added. The cooled mixture was stirredfor a further 1.5 hours. The mixture was diluted with ethyl acetate(100ml) and water (10ml) and sulphur dioxide was passed until themanganese dioxide had dissolved. The organic layer was separated andwashed successively with sodium bicarbonate solution, brine,N.hydrochloric acid, and brine The dried (MgSO₄) organic layer wasevaporated to give a crude gum (4.8g). The crude gum was chromatographedon silica gel eluting with ethyl acetate/petroleum ether mixtures togive 4-(4-phenylbut-2-ynylthio)-3-triphenylmethylaminoazetidin-2-one asa crystalline solid (0.852g, 22%). Recrystallisation of the product gavea colourless crystalline solid, MP=145°-6°C.

v_(max) (CHCl₃) 3400 cm⁻ ¹ (N-H), 1765 cm⁻ ¹ (β-lactam) δ ppm (CDCl₃)3.05 (centre of multiplet, 3H collapsing to triplet, 2H, J=2Hz with D₂O); 3.57 (t, 2H, J=2Hz); 4.55 (centre of multiplet, 2H, collapsing tobroad singlet with D₂ O); 6.15 (broad S, 1H, exchanges with D₂ O);7.0-7.7 (aromatics).

iii. Tert-butyl glyoxalate mono hydrate (2.1g) and dry benzene (25ml)were refluxed under nitrogen with provision for removal of water untilall the water had been removed.4-(4-phenylbut-2-ynylthio)-3-(triphenylmethylamino)azetidin-2-one(0.700g) was added and the mixture refluxed under nitrogen for a further2 hours. The reaction mixture was cooled and washed with water (5 ×15ml). The dried (MgSO₄) organic layer was evaporated to give a gum. Thegum was chromatographed on silica gel eluting with ethylacetate/petroleum ether mixtures to give1-(1-hydroxy-1-tert-butoxycarbonylmethyl)-4-(4-phenylbut-2-ynylthio)-3-triphenylmethylamino-azetidin-2-oneas a solid foam (0.490g, 55%).

v_(max) (CHCl₃) 1765 cm⁻ ¹ (β-lactam); 1730 (ester).

iv.1-(1-Hydroxy-1-t-butoxycarbonylmethyl)-4-(4-phenylbut-2-ynylthio)-3-(triphenylmethylamino)azetidin-2-one(0.100 g) was dissolved in a mixture of dry tetrahydrofuran (1ml) anddry dioxan (1ml) and the resulting solution was cooled to -5° C to-10°C. Dry pyridine (0.038g) in dry dioxan (0.5ml) was added followed bypurified thionyl chloride (0.058g) in dry dioxan (0.5ml) dropwise in 3minutes. The resulting mixture was stirred at -5°C for 1 hour. Themixture was filtered and the residue washed with dry toluene (2ml). Thecombined filtrates were evaporated and the residual gum extracted withdry toluene (4 × 5ml). The combined extracts were filtered andevaporated to give a gum. Re-evaporation of the gum from dry ether gave1-(1-chloro-1-tert-butoxycarbonylmethyl)-4-(4-phenylbut-2-ynylthio)-3-triphenylmethylamino-azetidin-2-oneas a solid foam (99mg, 96%).

v_(max) (CHCl₃) 1775 cm⁻ ¹ (β-lactam); 1745 (ester.)

v. m-Chloroperbenzoic acid (30mg) in ethanol-free chloroform (3ml) wasadded in 10 minutes to a stirred solution of1-(1-chloro-1-t-butoxycarbonylmethyl)-4-(4-phenylbut-2-ynylthio)-3-(triphenylmethylamino)azetidin-2-one(99mg) in ethanol-free chloroform (3ml) at 0°C. The mixture was stirredfor a further 30 minutes at 0°C. The reaction mixture was diluted withethanol-free chloroform (10ml) and washed successively with saturatedsodium bicarbonate solution (5ml) and brine (2 × 5ml). The dried (MgSO₄)organic layer was evaporated to give a gum which upon re-evaporationfrom dry ether gave1-(1-chloro-1-tert-butoxycarbonylmethyl)-4-(4-phenylbut-2-ynylsulphinyl)-3-triphenylmethylamino-azetidin-2-oneas a solid foam (92mg, 91%)

v_(max) (CHCl₃) 1785 cm⁻ ¹ (β-lactam); 1740 (ester).

vi. Method A

The chloro-sulphoxide from (v) above, (46mg), triphenylphosphine (37mg)and 2,6-dimethylpyridine (9mg) were stirred and heated at 50°C in a drydioxan (1ml) under nitrogen for 12 hours. The mixture was diluted withethyl acetate (20ml) and washed successively with N-hydrochloric acid(5ml), and brine (2 × 5ml). The dried (MgSO₄) organic layer wasevaporated to give a gum. The gum was chromatographed on silica geleluting with ethyl acetate/petroleum ether mixtures to give4-(4-phenylbut-2-ynylsulphinyl)-1-(1-tert-butoxycarbonyl-1-triphenylphosphoranylidenemethyl)-3-triphenylmethylamino-azetidin-2-oneas a solid (9mg, 15%)

v_(max) (CHCl₃) 1765 cm⁻ ¹ (β-lactam); 1635 cm⁻ ¹.

Method B

The chloro-sulphoxide from (v) above (50mg), triphenylphosphine (40mg)and 1,8-bis(dimethylamino)naphthalene (16mg) were stirred and heated at50°C in dry dioxan (1ml) under nitrogen for 36 hours. The mixture wasevaporated to give a gum which gave, after chromatography as in MethodA, the desired phosphorane as a solid (7mg, 10%).

Method C

The chloro-sulphoxide from (v) above, (54mg), anhydrous lithium bromide(36mg) and triphenylphosphine (44mg) were stirred in dry tetrahydrofuran(2ml) at room temperature for 60 hours. Pyridine (2 drops) was added andthe mixture was stirred for a further 10 minutes. The mixture wasevaporated to give a gum which gave, after chromatography, as in MethodA, the desired phosphorane as a solid (13mg, 18%).

Method D

The chloro-sulphoxide from (v) above, (1.14g), anhydrous lithium bromide(0.76g) and triphenylphosphine (0.92g) were stirred and heated at 60°Cin dry tetrahydrofuran (30ml) under nitrogen for 2 hours. The mixturewas cooled to room temperature and pyridine (0.14g) was added. Themixture was stirred at room temperature for 10 minutes. The work-up asin method A, gave the desired phosphorane as a solid (0.244g, 17%).

vii.4-(4-phenylbut-2-ynylsulphinyl)-1-(1-t-butoxycarbonyl-1-triphenylphosphoranylidenemethyl)-3-(triphenylmethylamino)azetidin-2-one(244mg) was stood in piperidine (5ml) at room temperature for 24 hours.The mixture was diluted with ethyl acetate (100ml) and washed withN-hydrochloric acid (3 × 20ml) followed by brine. The dried (MgSO₄)organic layer was evaporated to give a gum (240mg). The gum waschromatographed on silica gel eluting with ethyl acetate/petroleum ethermixtures to give4-(4-phenyl-2-oxobutylsulphinyl)-1-(1-tert-butoxycarbonyl-1-triphenylphosphoranylidenemethyl)-3-triphenylmethylaminoazetidin-2-oneas a solid foam (204mg, 82%).

v_(max) (CHCl₃) 1770 cm⁻ ¹ (β-lactam); 1715 cm⁻ ¹ (ester + ketone); 1635cm⁻ ¹.

Preparation of starting materials for Example 2

i. Benzyl 6-β-(triphenylmethylamino)penicillanate (2.74g) was stirred indry tetrahydrofuran (50ml) containing 1-bromo-3-phenylprop-2-yne (1g)under nitrogen. Sodium hydride (0.48 g of 60% oil dispersion) was addedand the mixture was stirred at room temperature for 48 hours. Thereaction mixture was then diluted with ethyl acetate and the organiclayer washed with brine and water. The dried ethyl acetate extract wasevaporated to dryness and the residue triturated with ethyl acetate.Filtration gave unchanged starting material (1.05g). Chromatography ofthe mother liquors on silica, eluting with ethyl acetate/petroleum ether(1:9), afforded further unchanged starting ester (230mg) and1-(1-benzyloxycarbonyl-2-methyl-1-propanyl)-3-(triphenylmethylamino)-4-(3-phenylprop-2-ynylthio)azetidin-2-one,as a foam (905mg).

v_(max) (CHCl₃) 1760 (β-lactam, 1715 (ester), 1625 (double bond.

δ ppm (CDCl₃) 2.07 (s, 3H), 2.17 (s, 3H), 2.95 (AB quartet, J=17Hz),2.95 (b, 1H, exchanged by D₂ O), 4.55 (broadened signal collapsing to adoublet, 1H, J=5Hz, after D₂ O exchange), 4.93 (d, J=5Hz), 4.98 (s, 2H),7-7.7 (m, 25H).

ii.1-(1-Benzyloxycarbonyl-2-methyl-1-propenyl)-3-(triphenylmethylamino)-4-(3-phenylprop-2-ynylthio)azetidin-2-one (I) (3.24g) was dissolved in pyridine (30ml) and water(2ml) and the mixture was cooled in an ice-bath. Solid potassiumpermanganate (1.19g) was added, and the mixture stirred for 1 hour.Ethyl acetate and brine were added and the mixture vigorously shaken tocoagulate the manganese dioxide. The latter was removed by filteringthrough kieselguhr, the filter cake being washed well with ethylacetate. The organic layer was separated, washed with N-hydrochloricacid and water, dried, and evaporated to a foam (2.67g). Chromatographyon silica gave unchanged starting material (826mg) and3-(triphenylmethylamino)-4-(3-phenylprop-2-ynylthio) azetidin-2-one, asa foam (674mg). Trituration of the latter with 10% ethyl acetate/60°-80° petroleum ether gave a white solid (576mg). A samplerecrystallised from ethyl acetate/60°-80° petroleum ether had m.p.109°-110°.

v_(max) (CHCl₃) 3300, 3230, 1765 cm⁻ ¹.

iii. 3-(Triphenylmethylamino)-4-(3-phenylprop-2-ynylthio) azetidin-2-one(526mg) and methyl glyoxylate (1.17g) were refluxed in dry benzene(25ml) with provision for the removal of water. After 11/2 hours thesolvent was evaporated and the residue was chromatographed on silica togive 1-(1-hydroxy-1-methoxycarbonylmethyl)-3-(triphenylmethylamino)-4-(3-phenylprop-2-ynylthio)azetidin-2-one as an amorphoussolid (399 mg).

v_(max) (CHCl₃) 3475 (-OH), 1770 (β-lactam), 1750 (ester) cm⁻ ¹.

iv.1-(1-Hydroxy-1-methoxycarbonylmethyl)-3-(triphenylmethylamino)-4-(3-phenylprop-2-ynylthio)azetidin-2-one(395mg) was dissolved in a 1:1 mixture of dry tetrahydrofuran and dioxan(14ml) and the solution, under nitrogen, was cooled to -10°. Pyridine(176mg) in dry dioxan (1ml) was then added, followed by the dropwiseaddition of thionyl chloride (0.153ml) in dry dioxan (4ml) over 2-3minutes. After a further 15 minutes the precipitated solid was filteredoff and the filtrate evaporated to dryness. Dry toluene was added andwas decanted from any solid. The organic solution was evaporated to give1-(1-Chloro-1-methoxycarbonylmethyl)-3-(triphenylmethylamino)-4-(3-phenylprop-2-ynylthio)azetidin-2-oneas an amorphous solid (419mg), after drying overnight in vacuo.

v_(max) (CHCl₃) 1770 (broad, β-lactam and ester) cm⁻ ¹.

v.1-(1-Chloro-1-methoxycarbonylmethyl)-3-(triphenylmethylamino)-4-(3-phenylprop-2-ynylthio)azetidin-2-one(419mg) was dissolved in a 1:1 mixture of dry tetrahydrofuran and dioxan(12ml) under nitrogen. Triphenylphosphine (370mg) and dry pyridine(111mg) were added and the mixture heated at 55° for 13 hours. Thereaction mixture was filtered and the filtrate evaporated. The residuewas taken up in dry toluene and re-evaporated. Chromatography on silicaafforded1-(-11-(1-methoxycarbonyl-1-triphenylphosphoranylidenemethyl)-3-(triphenylmethylamino)-4-(3-phenylprop-2-ynylthio)azetidin-2-oneas a white solid (419mg).

v_(max) (CHCl₃) 1750 (broad), 1615 (broad) cm⁻ ¹.

vi.1-(1-Methoxycarbonyl-1-triphenylphosphoranylidenemethyl)-3-(triphenylmethylamino)-4-(3-phenylprop-2-ynylthio)azetidin-2-one(346mg) was refluxed in piperidine (8ml) containing mercuric chloride(242mg) for 8.5 hours. The mixture was stirred at room temperature for16 hours and then filtered through celite, the filter cake being washedwell with ethyl acetate and water. The organic layer was washed withdilute hydrochloric acid and brine, dried and evaporated to an amorphoussolid. Chromatography on silica afforded1-(1-methoxycarbonyl-1-triphenylphosphoranylidenemethyl)-3-(triphenylmethylamino)-4-(3-phenyl-prop-2-onethio)azetidin-2-oneas a white solid (245 mg).

v_(max) (CHCl₃) 1775 (broad), 1720 (broad), 1615 (broad) cm⁻ ¹.

Preparation of starting materials for Example 3

The procedure used for the preparation of starting materials for Example2 was followed, except that t-butyl glyoxalate was used in stage (iii)instead of methyl glyoxalate. Eventually1-(1-t-butoxycarbonyl-1-triphenylphosphoranylidenemethyl)-3-(triphenylmethylamino)-4-(3-phenyl-2-oxopropylthio)azetidin-2-onewas prepared.

v_(max) (CHCl₃) 1755, 1720, 1635 cm⁻ ¹.

Preparation of starting materials for Example 4

i. Benzyl 6-β-(triphenylmethylamino)penicillanate (6.1g) was stirred indry tetrahydrofuran (100ml) under nitrogen and1-bromo-3-p-fluorophenylprop-2-yne (3.04g) was added. Potassiumt-butoxide (11.1ml of a 1.1M solution in tertiary butanol, diluted with10ml dry tetrahydrofuran) was added dropwise over 2.5 hrs. Afterstirring for a further 2.5 hours the mixture was diluted with ethylacetate and the organic layer washed with brine, dried and evaporated.On trituration with ethyl acetate unchanged starting material (0.81g)was obtained as white crystals. Chromatography of the mother liquors onsilica, eluting with ethyl acetate/petroleum ether (1.9) affordedfurther unchanged starting ester (0.525g) and1-(1-benzyloxycarbonyl-2-methyl-1-propanyl)-3-(triphenylmethylamino)-4-(3-p-fluorophenylprop-2-ynylthio)azetidin-2-one(3.78g) which was recrystallised from ethyl acetate/petroleum ether togive white crystals m.p. 123°-4°.

v_(max) (CHCl₃). 1758 (β-lactam), 1718 (ester), 1625 (double bond)cm.sup.⁻¹.

δ ppm (CDCl₃). 2.02 (s, 3H); 2.17 (s, 3H); 2.94 (q, 2H, J= 16Hz,covering 1H exch. D₂ O); 4.54 (m, 1H, collapsing to d, J=5Hz on D₂ Oexchange); 4.9 (d, 1H, J=5Hz); 4.97 (q, 2H, J=12Hz); 6.8-7.6 (24 ar).

ii.1(1-Benzyloxycarbonyl-2-methyl-1-propenyl)-3-(triphenylmethylamino)-4-(3-p-fluorophenylprop-2-ynylthio)azetidin-2-one (2.68g) was dissolved in pyridine (30ml) and water (2ml).The mixture was cooled in an ice bath and potassium permanganate (0.93g)added with stirring. After one hour ethyl acetate (50ml) and water (5ml)were added and sulphur dioxide was passed into the cooled mixture untilit became colourless. The organic layer was separated, washed withsaturated sodium bicarbonate solution, brine, N-hydrochloric acid andfinally brine, dried and evaporated to a foam. Chromatography on silica-H gave unchanged starting material (0.77g) and3-(triphenylmethylamino)-4-(3-p-fluorophenylprop-2-ynylthio)azetidin-2-oneas a foam (0.71g).

v _(max) (CHCl₃) 3350, 1765 cm.sup.⁻¹.

δ ppm (CDCl₃) 2.94-3.2 (1H, exchange D₂ O),3.25 (s, 2H), 4.58 (2H broadpeak sharpening on D₂ O exchange), 6.22 (s, 1H exchange D₂ O), 6.81-7.58(Aromatic).

iii.3-(Triphenylmethylamino)-4-(3-p-fluorophenylprop-2-ynylthio)azetidin-2-one(1.02g) and t-butyl glyoxalate (2.76g) were refluxed in dry benzene(50ml) with provision for the removal of water. After 1 hour the benzenesolution was washed 5 times with water, dried and evaporated. Theresidue was chromatographed on silica to give1-(1-Hydroxy-1-t-butoxycarbonylmethyl)-3-(triphenylmethylamino)-4-(3-p-fluorophenylprop-2-ynylthio)azetidin-2-oneas an amorphous solid (.969g).

v _(max) (CHCl₃) 1770, 1735 cm.sup.⁻¹.

iv. 1(1-Hydroxy-1-t-butoxycarbonylmethyl)-3-(triphenylmethylamino)-4-(3-p-fluorophenylprop-2-ynylthio)azetidin-2-one(570mg) was dissolved in a 1:1 mixture of dry tetrahydrofuran and dioxan(20ml) and the solution, under nitrogen, was cooled to -10°. Pyridine(224mg) in dry dioxan (5ml) was then added, followed by the dropwiseaddition of thionyl chloride (0.206ml) in dry dioxan (5ml) over 2-3minutes. After a further 15 minutes the precipitated solid was filteredoff and the filtrate evaporated to dryness. Dry toluene was added andwas decanted from any solid. The organic solution was evaporated todryness and re-evaporation from dry ether gave1-(1-Chloro-1-t-butoxycarbonylmethyl)-3-(triphenylmethylamino)-4-(3-p-fluorophenylprop-2-ynylthio)azetidin-2-oneas an amorphous solid.

v.1-(1-Chloro-1-t-butoxycarbonylmethyl)-3-(triphenylmethylamino)-4-(3-p-fluorophenylprop-2-ynylthio)azetidin-2-one(570mg) was dissolved in a 1:1 mixture of dry tetrahydrofuran and dioxan(20ml) under nitrogen. Triphenylphosphine (495mg) and dry pyridine(150mg) were added and the mixture heated at 49° for 16 hours. Thereaction mixture was filtered the filtrate evaporated. The residue wastriturated with toluene and the soluble portion re-evaporated.Chromatography on silica afforded1-(1-t-butoxycarbonyl-1-triphenylphosphoranylidenemethyl)-3-(triphenylmethylamino)-4-(3-p-fluorophenylprop-2-ynylthio)azetidin-2-one, as a white solid (418mg).

v _(max) (CHCl₃) 1745, 1630 (broad) cm.sup.⁻¹.

vi.1-(1-t-Butoxycarbonyl-1-triphenylphosphoranylidenemethyl)-3-(triphenylmethylamino)-4-(3-p-fluorophenylprop-2-ynylthio)azetidin-2-one(370mg) was refluxed is piperidine (25ml) under nitrogen for 6 hours.The mixture was cooled, diluted with ethyl acetate and the organic layerwashed with N-hydrochloric acid and brine, dried and evaporated to anamorphous solid. Chromatography on silica gave1-(1-t-butoxycarbonyl-1-triphenylphosphoranylidenemethyl-3-(triphenylmethylamino)-4-(3-p-fluorophenyl-2-oxopropylthio)azetidin-2-oneas an amorphous solid (330mg).

v _(max) (CHCl₃) 1750, 1718, 1625 (broad) cm.sup.⁻¹. 0.25

Preparation of starting materials for Example 5

i. Benzyl 6-β-(triphenylmethylamino)penicillanate (1.1g) in drytetrahydrofuran (40ml) containing 1-bromobut-2-yne (0.3g) (under N₂) wastreated with sodium hydride (0.2g of 50% dispersion) and refluxed for 7hours and then left stirring overnight at room temperature. The reactionmixture was diluted with ethyl acetate (150ml) and washed with brine andwater. The dried ethyl acetate extract was evaporated to dryness and theresidue triturated with ethyl acetate. Filtration gave unchanged (I)(0.5g). Chromatography of the mother liquors on silica, eluting withethyl acetate light petroleum (3:7) gave more unchanged startingmaterial (0.11g) and then1-(1-benzyloxycarbonyl-2-methyl-1-propenyl)-3-(triphenylmethylamino)-4-(but-2-ynylthio)azetidin-2-oneas a foam (0.25g).

v _(max) (CHCl₃) 1760 cm.sup.⁻¹ (β-lactam carbonyl), 1720 cm.sup.⁻¹(ester), 1625 cm.sup.⁻¹ (C = C)

δ ppm,(CDCl₃) 1.67 (t, 3H, J=2.5Hz); 2.00 (s, 3H); 2.22 (s, 3H); 2.63(q, 2H, J=2.5Hz);2.92 (d, 1H, exch. NH); 4.50 (dd, 1H, collapsing tosinglet J=5H D₂ O exchange 4.75 (d, 1H, J=5Hz); 5.08 (q, 2H, J=12Hz);7.1-7.6 (Ar, 20H).

ii.1-(1-Benzyloxycarbonyl-2-methyl-1-propenyl)-3-(triphenylmethylamino)-4-(but-2-ynylthio)azetidin-2-one(4.4g) was dissolved in dimethylformamide (40ml), water (4ml) andpyridine (1.6ml) and the mixture cooled to -20°. Solid potassiumpermanganate (1.74g) was added, and the mixture stirred between -20° and-5° for 2 hours. Ether and water were then added and the mixture shaken.After filtering through Keiselguhr, the organic layer was washed with Nhydrochloric acid and brine, dried and evaporated to give an amorphoussolid (2.73g). Chromatography on silica -H gave unchanged startingmaterial (0.7g) and 3-(triphenylmethylamino)-4-(but-2-ynylthio)azetidin-2-one (1.2g) as a foam.

v _(max) (CHCl₃) 3370, 1770 cm.sup.⁻¹.

δ ppm (CDCl₃) 1.73 (t, 3H, J=3Hz), 2.97 (q, 2H, J=3Hz), 4.50 (broadenedsignal 2H, collapsing to sharp singlet on D₂ O exchange), 6.54 (b.s.,1H, exchange), 7.1-7.7 (Ar, 15H).

iii. 3-(Triphenylmethylamino)-4-(but-2-ynylthio)azetidin-2-one (1.03g)and t-butyl glyoxalate (3.3 g) were refluxed in dry benzene (50ml) withprovision for the removal of water. After one hour the benzene solutionwas washed 5 times with water, dried and evaporated. Chromatography ofthe residue on silica -H gave1-(1-hydroxy-1-t-butoxycarbonylmethyl)-3-(triphenylmethylamino)-4-(but-2-ynylthio)azetidin-2-one as an amorphous white solid (0.978 g)

v_(max) (CHCl₃) 3400, 1765, 1730 cm.sup.⁻¹.

iv.1-(1-Hydroxy-1-t-butoxycarbonylmethyl)-3-(triphenylmethylamino)-4-(but-2-ynylthio)azetidin-2-one(0.44g) was dissolved in a 1:1 mixture of dry tetrahydrofuran and dioxan(10ml) and the solution, under nitrogen, was cooled to -10°. Pyridine(0.2g) in dry dioxan (2.5ml) was added, followed by the dropwiseaddition of thionyl chloride (0.19ml) in dioxan (2.5ml) over 2-3minutes. After a further 15 minutes the precipitated solid was filteredoff and the filtrate evaporated to dryness. Dry toluene was added to theresidue and decanted off from any solid. The organic solution wasevaporated to dryness and re-evaporation from dry ether gave1-(1-chloro-1-t-butoxycarbonylmethyl)-3-(triphenylmethylamino)-4-(but-2-ynylthio)azetidin-2-oneas an amorphous solid (0.45g).

v _(max) (CHCl₃) 1775, 1740 cm.sup.⁻¹.

This chloride, without further purification was dissolved inethanol-free chloroform and the solution, under nitrogen, was cooled inan ice-bath. m-chloroperbenzoic acid (0.16g) in chloroform was addeddropwise over a few minutes. After a further 30 minutes the organicsolution was washed with saturated aqueous sodium bicarbonate, then withbrine, and finally dried and evaporated. Re-evaporation of the residuefrom dry ether gave1-(1-chloro-1-t-butoxycarbonylmethyl)-3-(triphenylmethylamino)-4-(but-2-ynylthio)azetidin-2-one sulphoxide (as an amorphous solid).

v _(max) (CHCl₃) 1785, 1740 cm.sup.⁻¹.

This sulphoxide, without further purification, was treated undernitrogen with triphenylphosphine (0.52g) and 2, 6-dimethylpyridine(0.125g) in dry dioxan (10ml) at 50° for 17 hours. The mixturewas filtered and the filtrate evaporated to dryness. Chromatography onsilica -H gave1-(1-t-butoxycarbonyl-1-triphenylphosphoranylidenemethyl)-3-(triphenylmethylamino)-4-(but-2-ynylthio)azetidin-2-onesulphoxide (0.2g) as a white solid.

v _(max) (CHCl₃) 1760 (b), 1730 (b) cm.sup.⁻¹.

v. 1-(1-t-Butoxycarbonyl-1-triphenylphosphoranylidenemethyl)-3-(triphenylmethylamino)-4-(but-2-ynylthio) azetidin-2-onesulphoxide (0.39g) was treated with piperidine (40ml) at roomtemperature for 24 hours. Ethyl acetate was added and the organic phasewashed with N-hydrochloric acid (x 3) and brine, dried and evaporated.Chromatography on silica -H gave1-(1-t-butoxycarbonyl-1-triphenylphosphoranylidenemethyl)-3-(triphenylmethylamino)-4-(2-oxobutylthio)azetidin-2-onesulphoxide (0.27g), obtained as a white solid.

v _(max) (CHCl₃) 1765, 1710, 1635 (b) cm.sup.⁻¹.

Preparation of starting materials for Example 6

i. Benzyl 6-β-(triphenylmethylamino)penicillanate (7.17g) was stirred indry tetrahydrofuran (90ml) containing1-bromo-4-tetrahydropyranyloxy-but-2-yne(3.1g) under nitrogen, and asolution of potassium t-butoxide (1.4g) in t-butanol (12.5ml) in drytetrahydrofuran (10ml) was added dropwise over 30 minutes. After sirringfor a further 1 hour the reaction mixture was worked-up as before. Thecrude product was chromatographed on silica (100g) eluting with ethylacetate/petroleum ether (1:9) to give1-(1-benzyloxycarbonyl-2-methyl-1-propenyl)-3-(triphenylmethylamino)-4-(4-tetrahydropyranyloxybut-2-ynylthio)azetidin-2-oneas an amorphous solid (4.05g).

v _(max) (CHCl₃) 1755 (β-lactam), 1715 (ester) 1620 (double bond)cm.sup.⁻¹.

δ ppm (CDCl₃) 1.6 (m, 6H), 1.98 (s, 3H), 2.21 (s, 3H), 2.72 (2H, ABquartet J=15Hz each signal being further split with J=1.5Hz), 2.9 (m,1H, D₂ O exchanged), 3.55 (m, 1H), 4.98 (t, 2H, J=1.5Hz), 4.5 (m, 1H,collapsing to doublet J=5Hz on D₂ O exchange), 4.75 (d, 1H, J=5Hz), 5.07(q, 2H, J=12Hz), 7.1-7.6 (m, 20H).

ii.1-(1-Benzyloxycarbonyl-2-methyl-1-propenyl)-3-(triphenylmethylamino)-4-(4-tetrahydropyranyloxybut-2-ynylthio)azetidin-2-one(7g) in dimethylformamide (50ml) containing water (5ml) and pyridine(2ml) was cooled to -10° and powdered potassium permanganate (2.4g) wasadded all at once. After 1 hour at -10° the mixture was poured intoether, shaken with brine and filtered through kieselguhr. The etherlayer was separated, washed with a little dilute hydrochloric acidfollowed by brine, dried, and evaporated. Chromatography on silicaafforded unchanged starting material (2.03g) and the required product3-(triphenylmethylamino)-4-(4-tetrahydropyranyloxybut-2-ynylthio)azetidin-2-one(1.36g) as an amorphous solid.

v _(max) (CHCl.sub. 3) 3400, 3300, 1768 cm.sup.⁻¹.

δ ppm (CDCl₃) 1.63 (broad singlet, 6H), 3.17 (t, 2H, J=2Hz), 3.00 (m,1H, D₂ O exchanged), 3.7 (m, 3H), 4.23 (t, 2H, J=2Hz), 4.63 (m, 2H), 6.7(broad singlet, 1H, D₂ O exchanged), 7.0-7.6 (m, 15H).

ii.3-(Triphenylmethylamino)-4-(4-tetrahydropyranyloxybut-2-ynylthio)azetidin-2-one(934mg) and t-butylglyoxylate (2.5g) were refluxed in benzene (20ml)with provision for the removal of water. After 1 hour the cooledsolution was washed with water (6 × 5ml), dried and evaporated.Chromatography on silica gave11-hydroxy-1-t-butoxycarbonylmethyl)-3-(triphenylmethylamino)-4-(4-tetrahydropyranyloxybut-2-ynylthio)azetidine-2-onewhich still contained some t-butylglyoxylate. Re-chromatography onsilica afforded the pure material as an amorphous solid (519 mg)

v_(max) (CHCl₃) 3500, 3350, 1770, 1738 cm.sup.⁻¹.

iv. 1-(1-Hydroxy-1-t-butoxycarbonylmethyl)-3-(triphenylmethylamino)-4-(4-tetrahydropyranyloxybut-2-ynylthio)azetidin-2-one (646mg) was dissolved in dry THF/dioxan (1:1, 12ml) and thesolution, under nitrogen, was cooled to -15°. Dry pyridine (227mg) indioxan (1ml) was then added followed by the dropwise addition of thionylchloride (357mg) in 1:1 tetrahydrofuran/dioxan (5ml) over 1-2 minutes.After a further 15 minutes the precipitated solid was filtered off andthe filtrate evaporated to dryness. Dry toluene was added and thesolution decanted from any solid, and evaporated to give1-(1-chloro-1-t-butoxycarbonylmethyl)-3-(triphenylmethylamino)-4-(4-tetrahydropyranyloxybut-2-ynylthio)azetidine-2-oneas an amorphous solid (650mg) after drying overnight in vacuo.

v _(max) (CHCl₃) 1780, 1745 cm.sup.⁻¹.

v.1-(1-chloro-1-t-butoxycarbonylmethyl)-3-(triphenylmethylamino)-4-(4-tetrahydropyranyloxybut-2-ynylthio)azetidin-2-one (650mg) was dissolved in 1:1 THF/dioxan (12ml) undernitrogen. Triphenylphosphine (525 mg) and pyridine (158 mg) were addedand the mixture heated at 55° for 15.5 hours. The reaction mixture wasfiltered and the filtrate evaporated. Dry toluene was added to theresidue and the solution decanted from any solid and evaporated.Chromatography on silica afforded1-(1-t-butoxycarbonyl-1-triphenylphosphoranylidenemethyl)-3-(triphenylmethylamino)-4-(4-tetrahydropyranyloxybut-2-ynylthio)azetidin-2-oneas an amorphous solid (470mg).

v_(max) (CHCl₃) 1755, 1638 cm.sup.⁻¹.

vi.1-(1-t-Butoxycarbonyl-1-triphenylphosphoranylidenemethyl)-3-(triphenylmethylamino)-4-(4-tetrahydropyranyloxybut-2-ynylthio)azetidin-2-one(462mg) in piperidine (7ml) was refluxed under nitrogen for 17 1/2hours. The solvent was removed under reduced pressure and the residuetaken up in ethyl acetate. The solution was washed with a little dilutehydrochloric acid followed by brine, dried and evaporated.Chromatography on silica afforded unchanged starting material (47mg)1-(1-t-butoxycarbonyl-1-triphenylphosphoranylidenemethyl)-3-(triphenyl-methylamino)-4-(4-tetrahydropyranyloxy-2-oxobutylthio)azetidin-2-oneas an amorphous solid (224 mg).

v _(max) (CHCl₃) 1778, 1720, 1638 cm.sup.⁻¹.

Preparation of starting material for Example 7

i. Benzyl 6-triphenylmethylamino-penicillanate (30g) was suspended indry tetrahydrofuran (500ml) under nitrogen and1-bromo-3-(2-tetrahydropyranyl)-prop-2-yne (11.7g) was added. A 0.78Msolution of potassium t-butoxide in t-butanol (78ml) was then addeddropwise under nitrogen to the stirred mixture over 3 hours. The mixturewas stirred under nitrogen for 1 hour after the complete addition of thebutoxide, then ethyl acetate was added and the solution was washed withbrine followed by water. The organic layer was dried (MgSO₄) andevaporated to leave an oil which was chromatographed on silica, elutingwith petroleum (b.p. 60°-80°)/ethyl acetate in 9:1, 17:3 and 8:2mixtures. The product1-(1-benzyloxycarbonyl-2-methyl-1-propenyl)-3-triphenylmethylamino-1-[3-(2-tetrahydropyranyl)-propynylthio]azetidin-2-onewas obtained as a foam (19g).

v _(max) (CHCl₃), 1755 cm.sup.⁻¹ (β-lactam carbonyl), 1718 cm.sup.⁻¹(ester), 1625 cm.sup.⁻¹ (C=C).

δppm (CDCl₃) 1.1-1.9 (broad s, 6H); 1.99 (s, 3H); 3.3-5.0 (complex, 5H);AB q. centred at 5.1 (2H), 7.1-7.2 (m, 20H).

ii.1-(1-Benzyloxycarbonyl-2-methyl-1-propenyl)-3-(triphenylmethylamino)-4-(3-(2-tetrahydropyranyl)-prop-2-ynylthio)]azetidin-2-one(2.2g) was dissolved in pyridine (20ml) and water (2ml) was added. Themixture was stirred and cooled in an ice-salt bath and finely groundpotassium permanganate (780 mg) was added. The mixture was stirred withcooling for 1 hour and then ethyl acetate/brine was added. Sulphurdioxide was passed into the mixture until all the manganese dioxide haddissolved, and then the layers were separated and the organic layer waswashed successively with aqueous sodium bicarbonate, N.HCl, and brine.The dried (MgSO₄) organic layer was evaporated to an oil andchromatographed on silica gel, eluting with ethyl acetate/petroleum(b.p. 60°-80°) in 2:8 and 3:7 mixtures, to give4[3-(2-tetrahydropyranyl)prop-2-ynylthio]-3-triphenylmethylamino-azetidin-2-one(340mg) in later fractions.

v _(max) (CHCl₃) 1765 cm.sup.⁻¹.

iii. Tert-butyglyoxylate hydrate (370mg) was refluxed in dry benzene(8ml) with provision for the removal of water present and then4[3-(2-tetrahydropyranyl)prop-2-ynylthio]-3-(triphenylmethylamino)azetidin-2-one(120mg) was added in benzene (4ml) and the mixture refluxed. After 23/4hours the mixture was cooled and benzene was added. The benzene solutionwas washed five times with water and once with brine, dried (MgSO₄) andevaporated to an oil which was chromatographed on silica gel, elutingwith ethyl acetate/petroleum (b.p. 60°-80°) in 2:8 and 7:3 mixtures.This led to the isolation of1-(1-hydroxy-1-tert-butoxycarbonylmethyl)-3-(triphenyl-methylamino)-4-[3-(2-tetrahydropyranyl)prop-2-ynylthio]-azetidin-2-one(90mg)

v _(max) (CHCl₃) 1775 cm.sup.⁻¹ (b-lactam C=O) and 1740 cm.sup.⁻¹ (esterC=O).

iv.1-(1-Hydroxy-1-t-butoxycarbonylmethyl)-3-(triphenylmethylamino)-4-[3-(2-tetrahydropyranyl)prop-2-ynylthio]azetidin-2-one (2.7g) was taken up in tetrahydrofuran (50ml) and thesolution was cooled to -15° and then pyridine (0.5ml) in tetrahydrofuran(15ml) were added to the cooled solution. The mixture was stirred at-15° for 45 minutes and then at room temperature for 15 minutes. Toluenewas then added and the precipitated pyridinium hydrochloride wasfiltered off. The solvent in the filtrate was removed to leave crude1-(1-t-butoxycarbonyl-1-chloromethyl)-3-(triphenylmethylamino)-4-[3-(2-tetrahydropyranyl)-prop-2-ynylthio]azetidin-2-oneas an oil.

v _(max) (CHCl₃) 1775 cm.sup.⁻¹ (β-lactam C=O) and 1745 cm.sup.⁻¹ (esterC=O). Tetrahydrofuran (30ml) and dioxan (25ml) were added to the crudechloride, the solution was warmed to 50°, and then triphenylphosphine(1.45g) and pyridine (0.5ml) were added. After stirring at 55° for 21hours, the solvents were removed and the crude product waschromatographed on silica gel, eluting with petroleum (b.p.60°-80°)/ethyl acetate (7:3). This gave1-(1-tert-butoxycarbonyl-1-triphenylphosphoranylidenemethyl)-3-(triphenylmethylamino)-1-[3-(2-tetrahydropyranyl)-prop-2-ynylthio]azetidin-2-oneas a glass (1.96g),

v _(max) (CHCl₃) 1750 cm.sup.⁻¹, 1635 cm.sup.⁻¹.

v.1-(1-t-Butoxycarbonyl-1-triphenylphosphoranyledene-methyl)-3-(triphenylmethylamino)-4-[3-(2-tetrahydropyranyl)-prop-2-ynylthio]azetidin-2-one,(1.86g) was taken up in pyrrolidine (15ml) and the mixture was refluxedunder nitrogen for 19 hours. The pyrrolidine was then removed and theresidue taken up in chloroform (150ml) and washed with 0.5N HCl (1 ×100ml, 2 × 50ml), followed by brine. The chloroform solution was thendried (MgSO₄) and evaporated to an oil, which as chromatographed onsilica gel (20g), using a gradient elution of petroleum (b.p.60°-80°)/ethyl acetate (from 7:3 to 1:1). This led to the isolation of1(1-tert-butoxycarbonyl-1-triphenylphosphoranylidenemethyl)-3-(triphenylmethylamino)-4-[3-(2-tetrahydropyranyl)-prop-2-onethio]azetidin-2-one(700mg)

v _(max) (CHCl₃) 1755; 1710, and 1635 cm.sup.⁻¹.

We claim:
 1. The compound methyl3-benzyl-7-triphenylmethylamino-3-cephem-4-carboxylate.